Production of hydroxyl radicals and alpha-dicarbonyl compounds associated with Amadori compound-Cu2+ complex degradation

The chemical properties of Amadori compounds in the presence of transition metal ions were studied, using the analogs 1-deoxy-1-n-butylamino-D-fructose (DBF) and N(alpha)-formyl-fructoselysine (fFL). The following characteristics were revealed: (a) DBF combined easily with Cu2+ (but no other transition metal ions) to form a DBF-Cu2+ complex in phosphate buffer, pH 7.4; (b) the complex was unstable, and degraded with the release of Cu+ during incubation at 37 degrees C; (c) degradation of the complex was associated with the production of hydroxyl radicals by the Fenton reaction and alpha-dicarbonyl compounds by non-autoxidative degradation; and (d) properties of DBF were similar to those of fFL. The above properties were additionally observed in glycated poly-Lys (GPL). Our findings indicate a novel mechanism for the generation of hydroxyl radicals and a-dicarbonyl compounds from Amadori adducts in the presence of Cu2+.     

Detection of underlying characteristics of nuclear chromatin patterns of thyroid tumor cells using texture and factor analyses

BACKGROUND: Aspiration biopsy cytology of thyroid tumors has been used more frequently in recent times to differentiate between malignant and benign lesions. Chromatin patterns of the tumor cell nuclei are one of most important factors for cytologic diagnosis. The interpretation of nuclear chromatin patterns is subjective and more difficult than that of nuclear size or shape. In the present report, we investigated how to detect underlying chromatin characteristics of benign and malignant thyroid tumor cells by means of texture and factor analyses. METHODS: We employed a computer-aided system in which light microscopy was combined with an image processor and monochrome camera. Using this system, 100 randomly selected cells in a Papanicolaou stained, aspiration biopsy cytologic smear in each case of 39 benign and malignant thyroid tumor cases were digitized. We applied two-dimensional and higher texture analyses with the use of co-occurrence and run-length matrices to analyze the chromatin patterns. Factor analysis was used to determine whether a large number of independent variables actually measured one or more underlying common variables. RESULTS: According to parameters with high factor-loading values, the morphologic chromatin characters were classified into three categories according to heterogeneity, contrast, and homogeneity of chromatin patterns. On the basis of analyses with these morphologic categories, nuclei of papillary carcinoma showed higher contrast of chromatin patterns than did those of the benign group. Moreover, there was a variety of contrasting chromatin patterns among cells in each papillary carcinoma case in comparison with the benign group. In contrast, follicular carcinomas showed a significant difference in the standard deviation of factor 3, which indicated more monotonous chromatin patterns among cells in each follicular carcinoma case than in each benign case. CONCLUSION: We believe that this technique, using texture and factor analyses, is useful in the detection of underlying characteristics of nuclear chromatin patterns in aspiration biopsy cytology.     

Sperm-egg binding mediated by sperm alpha-L-fucosidase in the ascidian,Halocynthia roretzi

Spermatozoa bind to the vitelline coat in the ascidians and many other animals. The binding of sperm in Halocynthia roretzi is mediated by a sperm alpha-L-fucosidase and complementary-L-fucosyl residues of glycoproteins in the vitelline coat. cDNA clones for alpha-L-fucosidase were isolated from growing testis mRNA. It contained a 1398 bp full-length cDNA insert (HrFuc'ase) that encoded the 466 amino acid residues of H. roretzi sperm alpha-L-fucosidase. A putative signal peptide of 21 amino acid residues proceeded the sequence for the mature protein (M.W. 52.4 kDa). The coding sequence for HrFuc'ase showed 47.7% sequence identity to the human liver fucosidase sequence. The polyclonal antibody was prepared against a lacZ-HrFuc'ase fusion protein expressed in E. coli. The antibody crossed to a 54 kDa protein in sperm on western blotting and inhibited fertilization in a dose dependent manner. These data suggest that sperm-egg binding is mediated by the sperm alpha-L-fucosidase, HrFuc'ase in the ascidian, H. roretzi.     

High levels of RAE-1 isoforms on mouse tumor cell lines assessed by anti-"pan" RAE-1 antibody confer tumor susceptibility to NK cells.

Two sublines of the benzpyrene-induced mouse hepatoma cell line, G-1 and G-5, showed low and high metastatic ability, respectively, to the lung. We produced a polyclonal antibody (pAb) against RAE-1alpha. Five isoforms of RAE-1 have been identified to date, and this pAb recognized all isoforms and was named anti-"pan" RAE-1 pAb. The level of RAE-1 was approximately 5-fold higher in G-5 than in G-1, which was almost RAE-1-negative, as determined using anti-pan RAE-1 pAb. Expression levels of other markers including MHC class I (MHC-I) and Qa-1b were very low and indistinguishable in these sublines. NK-mediated cytotoxicity was determined with these sublines; G-5 was highly susceptible to NK-mediated cytolysis, while G-1 was relatively resistant. The NK-mediated G-5 > G-1 killing profile was diminished if the G-5 cells were pretreated with F(ab)(2)(') of anti-pan RAE-1 pAb. G-1, when transfected with Rae-1alpha cDNA, acquired NK-responsiveness similar to that of G-5. These and additional data using mouse cell lines with low MHC-I levels and various RAE-1 levels also demonstrated that RAE-1 level is critically associated with NK-susceptibility in tumor cells.     

Early research on the role of plastocyanin in photosynthesis

A review is presented of the early history of investigations into the function of the blue copper-protein plastocyanin in photosynthesis. The controversy or confusion that arose as to the function of plastoycanin in conjunction with cytochrome f and cytochrome c ₆ is discussed and investigations contributing to the establishment of the role of plastocyanin as the mobile electron carrier between the Photosystem I reaction center complex and the cytochrome b ₆/f complex are described. This revised version was published online in August 2006 with corrections to the Cover Date.     

Diurnal and circadian rhythm in compound eye of cricket (Gryllus bimaculatus): changes in structure and photon capture efficiency

Day-night changes in rhabdom size of compound eyes were investigated in three groups of crickets (Gryllus bimaculatus): nymphs and adult males and females. In both adults and nymphs, the rhabdoms were larger at night than during a day. In adults, the mean rhabdom occupation ratios (RORs) of ommatidial retinulae at midnight were about two times greater than the values at midday. This change contributes to control of the photon capture efficiency (PCE) of the eye according to photic environment. The RORs of adult males at midnight were higher than those of both adult females and nymphs. This suggests that the PCE of the compound eye of adult males is the greatest of all groups. Under constant darkness, day-night changes in ROR were detected only in adult males, but neither in adult females nor in nymphs. On the other hand, no day-night changes were detected in any experimental group under constant light. These results suggest that the change in rhabdom size between day and night is an adaptation to the photic environment that is controlled mainly by the light-dark (day-night) cycle. However, the change in male adults is induced by an endogenous circadian clock.     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

Localization of sepiapterin reductase in pigment cells of Oryzias latipes

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.