We previously showed that serum-derived 85-kDa proteins (SHAPs, serum-derived hyaluronan associated proteins) are firmly bound to hyaluronan (HA) synthesized by cultured fibroblasts. SHAPs were then identified to be the heavy chains of inter-␣-trypsin inhibitor (ITI) (Huang, L., Yoneda, M., and Kimata, K. (1993) J. Biol. Chem. 268, 26725-26730). In this study, the SHAP⅐HA complex was isolated from pathological synovial fluid from human arthritis patients. The SHAP⅐HA complex was digested with thermolysin, followed by CsCl gradient centrifugation. The HA-containing fragments thus obtained were further digested with chondroitinase AC II and subjected to TSK gel high performance liquid chromatography (HPLC). Peptide-HA disaccharide-containing fractions (the SHAP⅐HA binding regions) were further purified by reverse phase HPLC. Major peaks were analyzed by protein sequencing and mass spectrometry (electrospray ionization mass spectrometry and collision induced dissociation-MS/MS). By comparison with the reported C-terminal sequences of the human ITI family, the peptides were found to correspond to tetrapeptides derived from the C termini of heavy chains 1 of and 2 of inter-␣-trypsin inhibitor (HC1 and HC2), and heavy chain 3 of pre-␣-trypsin inhibitor (HC3), respectively, and a heptapeptide from HC1. Mass spectrometric analyses suggested that the Cterminal Asp of each heavy chain was esterified to the C6-hydroxyl group of an internal N-acetylglucosamine of HA chain. This report is the first demonstration to give evidence for the covalent binding of proteins to HA.Hyaluronan (HA), 1 has been found as a ubiquitous component of the extracellular matrices of many tissues and in body fluids, including the vitreous body, synovial fluid, lymph, and blood (1-4). It has been suggested that HA plays an important role in many biological processes, such as gamete maturation, tissue morphogenesis, cell migration, and cell proliferation (5-8). HA is also involved in angiogenesis, wound healing, tumor invasion, and pathophysiological responses of tissues to inflammation (9 -12).With regard to functional importance, a large number of HA-binding proteins have been reported, an important subset of which have highly homologous sequences for HA binding. These include link proteins (13), hyaluronectin (14), glial HAbinding protein (15), HA-binding proteoglycan such as aggrecan, PG-M/versican (16), and CD44 (17). These are the proteoglycan tandem repeat families of HA-binding proteins. CD44 is a typical example of the family. Variant forms of CD44 generated by alternative splicing may have individual functions such as lymphocyte homing and tumor cell metastasis (18,19). Tumor necrosis factor-stimulated gene-6, another new member of this family, is tumor necrosis factor or interleukin-1-inducible and was recently shown to bind covalently to inter-␣-trypsin inhibitor (ITI) (20).We previously showed that serum-derived HA-associated proteins (SHAPs) appear to bind covalently to HA (21), and therefore to mediate the binding of HA to cell surfa...

Fetal DNA in maternal plasma and serum has been shown to be a useful material for fetal gender determination and for screening tests for abnormal pregnancies except during early gestational ages. Maternal serum samples were obtained from 81 pregnant women during the 5th-10th weeks of gestation. Fetal gender was determined by conventional polymerase chain reaction (PCR) to detect a Y-chromosomal sequence (DYS14) in maternal serum during early gestation and confirmed by examination of the newborns after delivery. Real-time quantitative analyses of the SRY and beta-globin genes were also performed in order to determine fetal gender and to quantify fetal DNA concentration in maternal serum during early gestation. When using conventional PCR, the total sensitivity of identifying a male fetus was 95%, but its sensitivity after the 7th week was 100%, whereas in real-time quantitative PCR, the total sensitivity after the 5th week was 100%. Quantitative analyses of the SRY gene revealed that the mean concentration of fetal DNA in maternal serum was 30.55 copies/ml, that fetal DNA concentration showed a tendency to increase with the progression of pregnancy, and that it had a wide normal range. Thus, we could confidently determine fetal gender by using maternal serum samples taken as early as the 7th week.

N-3 polyunsaturated fatty acids in fish oil exhibit a variety of health benefits, and there is evidence that they can inhibit the development of human lung mucoepidermoid and other carcinomas. To examine the hypothesis that fish consumption reduces the risk of lung cancer, we conducted a population-based prospective study, following 5,885 residents for 14 yr. Person-years were used to calculate the relative risk (RR) by the Cox proportional hazards model, with adjustment for potential confounding factors. A total of 51 incident lung cancer cases were observed, and we found linearly decreasing RRs for lung cancer with increased frequency of consumption of fish and shellfish (RRs = 1.00, 0.99, and 0.32, P for trend = 0.003) but not with intake of dried/salted fish. Decreased RRs were apparent with both broiling and boiling cooking methods, but reduction with raw and deep-fried fish consumption was not statistically significant. We conclude that frequent fresh fish consumption, irrespective of the cooking method, may reduce the risk of lung cancer.

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Frequencies of disomy and diploidy in spermatozoa for chromosomes X, Y and 18 were compared among severe oligozoospermic men (<5x10(6) spermatozoa/ml), oligozoospermic men (5-20x10(6) spermatozoa/ml), and normospermic men using three-colour fluorescence in-situ hybridization (FISH). Semen samples were collected from 10 severe oligozoospermic men aged 26-49 years, 10 oligozoospermic men aged 27-48 years and seven normospermic men aged 25-31 years. Karyotypes in lymphocytes obtained from peripheral blood were all 46,XY. In severe oligozoospermic men, analysis of 200 interphases per individual using FISH showed XY constitutions for sex chromosomes in all cells. A minimum of 10 000 sperm nuclei per individual for each chromosome was evaluated in severe oligozoospermic men and oligozoospermic men, and a minimum of 6000 sperm nuclei per individual in normospermic men. In total, 245 707 sperm nuclei were evaluated. The hybridization efficiency was 99.8%. The severe oligozoospermic men showed significantly higher frequencies of XY disomy (0.41%) and diploidy (0.49%) compared with oligozoospermic men (0.16%, P < 0.01; 0.22%, P < 0.05) and normospermic men (0.18%, P < 0.05; 0.21%, P < 0.05) (Mann-Whitney U-test). The data suggest that when severe oligozoospermic men undergo intracytoplasmic sperm injection, there can be an increase in the rate of conceptuses with 47,XXY chromosomes.