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This work describes the fabrication of quantum dot (QD)/silica (SiO2) core–shell particles immobilized with Au nanoparticles and the evaluation of their abilities in fluorescence and x-ray computed...

This work proposed a method for fabricating poly(lactic-co-glycolic acid) (PLGA) nanoparticles (ca. 70 nm) loaded with indocyanine green (ICG) molecules by an antisolvent crystallization method using dimethyl sulfoxide (DMSO) as a good solvent and water as a poor solvent. ICG molecules were loaded to PLGA nanoparticles by mixing PLGA, polyethylenimine, and polyethylene glycol (PEG) in DMSO with polyvinyl alcohol, albumin, and ICG in water. The ICG-loaded PLGA nanoparticle colloid solutions emitted fluorescence even at high ICG concentrations. Not only the tissues of a tumor-bearing mouse but also the tumor could be imaged with an IVIS system by injecting ICG-loaded PLGA nanoparticle colloid solution into the tail vein of the mouse and measuring the emitted fluorescence intensity, though some ICG-loaded PLGA nanoparticles were trapped in tissues such as the liver and spleen due to insufficient PEGylation. Thus, this work showed that ICG-loaded PLGA nanoparticles have potential for cancer imaging.

This work develops a process for fabricating particles composed of a quantum dot (QD) core, a SiO2 shell, and single-nanometer-sized Au nanoparticles (AuS) immobilized on the particle surface. The QD nanoparticles were silica-coated with a sol-gel process using tetraethyl orthosilicate (QD/SiO2). A colloidal solution of the AuS was prepared by reducing Au3+ with tetrakis(hydroxymethyl)phosphonium chloride in an aqueous solution. The QD/SiO2 particles were surface-modified with thiol groups by using (3-mercaptopropyl)trimethoxysilane (QD/SiO2/SH). The AuS nanoparticles were immobilized on the QD/SiO2/SH particle surface by adding a colloidal AuS solution to a colloidal QD/SiO2/SH solution (QD/SiO2/SH/AuS). The QD/SiO2/SH/AuS particles were surface-modified with thiol-terminated polyethylene glycol (PEG) (QD/SiO2/SH/AuS/PEG). Fluorescence images and computed tomography (CT) images could be obtained for the colloidal QD/SiO2/SH/AuS/PEG particle solution. The colloidal QD/SiO2/SH/AuS/PEG particle solution had a CT value per unit concentration of Au (M) of 6.68 × 103 HU/M. The circulation of the QD/SiO2/SH/AuS/PEG particles was confirmed in a mouse after injection of a colloidal particle solution into its blood vessels, and they accumulated in the heart. A portion of the QD/SiO2/SH/AuS/PEG particles accumulated in the liver and spleen and remained there.

This paper describes a method of coating metallic gold (Au) nanoparticles with chitosan (CS) shells in an aqueous solution. A colloidal solution of Au nanoparticles with a particle size of 16.5 ± 1.7 nm was prepared by reducing the Au ions (III) with sodium citrate in water at 80 °C. CS coating was achieved in an aqueous solution containing the Au nanoparticles by using CS as a shell source and sodium hydroxide as a pH adjuster at 25 °C. Ultraviolet-visible extinction spectroscopy and transmittance electron microscopy observation revealed that the Au nanoparticles were coated with CS shells. Additionally, the CS shell thickness could be varied in a range up to 2.2 nm by changing the CS concentration during the CS-coating process.

The technology to accurately image the morphology of tumor vessels with X-ray contrast agents is important to clarify mechanisms underlying tumor progression and evaluate the efficacy of chemotherapy. However, in clinical practice, iodine-based contrast agents present problems such as short blood retention owing to a high clearance ability and insufficient X-ray absorption capacity when compared with other high atomic number elements. To resolve these issues, gold nanoparticles (AuNPs), with a high atomic number, have attracted a great deal of attention as contrast agents for angiography, and have been employed in small animal models. Herein, we developed novel contrast agents using AuNPs and captured changes in tumor vessel morphology with time using X-ray computed tomography (CT). First, glutathione-supported single nanometer-sized AuNPs (sAu/GSH) (diameter, 2.2 nm) were fabricated using tetrakis(hydroxymethyl)phosphonium chloride as a reducing agent. The sAu/GSH particles were intravenously injected into mice, remained in vessels for a few minutes, and were then excreted by the kidneys after 24 h, similar to the commercial contrast agent iopamidol. Next, the Au/GSH and lactoferrin (sAu/GSH-LF) (long axis size, 17.3 nm) complex was produced by adding lactoferrin to the sAu/GSH solution under the influence of a condensing agent. On intravenously administering sAu/GSH-LF to mice, the blood retention time was 1-3 h, which was considerably longer than that observed with iopamidol and sAu/GSH. Moreover, we succeeded in imaging morphological changes in identical tumor vessels for several days using X-ray CT with sAu/GSH-LF.

Anticancer drug efficacy varies because the delivery of drugs within tumors and tumor responses are heterogeneous; however, these features are often more homogenous in vitro. This difference makes it difficult to accurately determine drug efficacy. Therefore, it is important to use living tumor tissues in preclinical trials to observe the heterogeneity in drug distribution and cell characteristics in tumors. In the present study, to accurately evaluate the efficacy of an antibody-drug conjugate (ADC) containing a microtubule inhibitor, we established a cell line that expresses a fusion of end-binding protein 1 and enhanced green fluorescent protein that serves as a microtubule plus-end-tracking protein allowing the visualization of microtubule dynamics. This cell line was xenografted into mice to create a model of living tumor tissue. The tumor cells possessed a greater number of microtubules with plus-ends, a greater number of meandering microtubules, and a slower rate of microtubule polymerization than the in vitro cells. In tumor tissues treated with fluorescent dye-labeled ADCs, heterogeneity was observed in the delivery of the drug to tumor cells, and microtubule dynamics were inhibited in a concentration-dependent manner. Moreover, a difference in drug sensitivity was observed between in vitro cells and tumor cells; compared with in vitro cells, tumor cells were more sensitive to changes in the concentration of the ADC. This study is the first to simultaneously evaluate the delivery and intracellular efficacy of ADCs in living tumor tissue. Accurate evaluation of the efficacy of ADCs is important for the development of effective anticancer drugs.

Previous studies showed that gold nanoparticles (AuNPs) are useful radiosensitizers which optimize radiation therapy under low-dose radiation. However, the mechanisms of AuNP radiosensitization, including the amount and localization of the AuNPs interacting with cancer cells, has not yet been quantified. To answer these questions, we prepared AuNPs conjugated with anti-human epidermal growth factor receptor type 2 (HER2) antibody via polyethylene glycol (PEG) chains (AuNP-PEG-HER2ab). AuNP-PEG-HER2ab specifically bound to the HER2-expressing cancer cells and entered the cells via endocytosis. Whether endocytosis of AuNP-PEG-HER2ab occurred had no effect on radiosensitization efficacy by AuNP-PEG-HER2ab in vitro. The radiosensitization efficacy in vitro depended on dose of AuNP-PEG-HER2ab or dose of X-ray. Moreover, AuNP-PEG-HER2ab administrated into tumor-bearing mice was localized to both the periphery of the tumor tissue and near the nuclei in cancer cells in tumor deep tissue. The localization of AuNP-PEG-HER2ab in tumor tissues was important factors for in vivo powerful radiosensitization efficacy.

This study reports two findings. The first is a method for preparing a colloidal solution of silica (SiO 2 ) nanoparticles activated with fluorescent dye or fluorescent dye-active SiO 2 nanoparticles. Preparation of a colloid solution of spherical SiO 2 nanoparticles with an average size of 109.4 ± 8.6 nm was performed using the sol-gel method at 35 °C using 0.2 M tetraethylorthosilicate, 25 M H 2 O, and 0.01 M NaOH in ethanol. Two kinds of cationic chemicals (poly-diallyldimethylammonium chloride (PDADMAC) and (3-aminopropyl) triethoxysilane (APES) were examined for surface-modification or surface-cationization of the SiO 2 nanoparticles (SiO 2 /PDADMAC and SiO 2 /APES). To activate the SiO 2 nanoparticles with fluorescent dye, indocyanine green (ICG) molecules were fixed on the surface-modified SiO 2 nanoparticles by mixing ICG dissolved in albumin aqueous solution and the surface-modified SiO 2 nanoparticle colloid solution (SiO 2 /PDADMAC/ICG and SiO 2 /APES/ICG) through electrostatic aggregation. The second finding is the verification of the fluorescence ability of the ICG-active SiO 2 nanoparticle colloid solutions. Both the ICG-active SiO 2 nanoparticle colloid solutions exerted fluorescence even at high ICG concentrations. Mouse tissues could be imaged by injecting the ICG-immobilized SiO 2 nanoparticle colloid solution into the tail vein of the mouse and measuring the emitted fluorescence intensity. Pulmonary embolism was avoided in the SiO 2 /APES/ICG particle colloid solution-injected group, in which the particles reached the tissues more efficiently than in the SiO 2 /PDADMAC/ICG nanoparticles-injected group.

BACKGROUND/AIM We have previously reported that treatment of pancreatic cancer cells with active hexose-correlated compound (AHCC), an extract of a basidiomycete mushroom, decreases the levels of tumor-associated proteins including heat-shock protein 27 (HSP27), heat shock factor 1 (HSF1) and sex-determining region Y-box 2 (SOX2). The transmembrane glycoprotein, CUB domain-containing protein 1 (CDCP1) has been reported to be up-regulated in various cancers, and be associated with invasion and metastasis. The aim of this study was to examine the effect of AHCC on the expression of CDCP1 in KLM1-R cells. MATERIALS AND METHODS Gemcitabine-resistant pancreatic cancer cells (KLM1-R) were treated with AHCC (10 mg/ml) for 48 h. Western blot analysis of cell extracts with anti-CDCP1 or anti-actin antibodies was performed to assess the expression of CDCP1. RESULTS Expression of CDCP1 was reduced by AHCC treatment of KLM1-R cells, whereas expression of actin was not affected. The ratio of intensities of CDCP1/actin in AHCC-treated KLM1-R cells was significantly suppressed (p

The present work proposes a preparation method for core-shell nanoparticles composed of quantum dots (QDs), SiO2, large Au nanoparticles (AuL), and small Au nanoparticles (AuS). The QDs wer...

This study described the preparation of silica-coated Au nanorods (AuNR/SiO2) in a colloidal solution, assessed their property of photothermal conversion, and investigated their ability to kill cancer cells using photothermal conversion. Au-seed nanoparticles were produced by reducing hydrogen tetrachloroaurate (HAuCl4) with sodium borohydride (NaBH4) in aqueous n-hexadecyltrimethylammonium bromide (CTAB) solution. AuNRs were then fabricated by reducing HAuCl4 and silver nitrate (AgNO3) with l-ascorbic acid in the aqueous CTAB solution in the presence of Au-seed nanoparticles. The as-prepared AuNRs were washed by a process composed mainly of centrifugation to remove the CTAB. The washed AuNRs were coated with silica by mixing the AuNR colloidal solution, an aqueous solution of (3-aminopropyl)trimethoxysilane, and tetraethylorthosilicate/ethanol solution with a water/ethanol solution. We found that the addition of AuNR/SiO2 in water, in mice, and in a culture medium with cancer cells, followed by irradiation with a laser, cause an increase in temperature, demonstrating that AuNR/SiO2 have the ability of photothermal conversion. In addition, the cancer cells in the culture medium were found to be killed due to the increase in temperature caused by the photothermal conversion.

The present work proposes a preparation method for multilayered Au nanoparticle/silica/gadolinium compound core–shell (Au/SiO2/GdC) particles. Silica-coated Au core–shell (Au/SiO2) particles with a size of 38.0 nm were prepared by a sol-gel reaction in the presence of the Au nanoparticles with a size of 15.5 nm. Multilayered Au/SiO2/GdC particles with sizes of ca. 35–52 nm were prepared by a homogeneous precipitation reaction in the presence of Au/SiO2 particles. The computed tomography (CT) value of the Au/SiO2/GdC colloid solution containing 4.3 × 10−2 M Au was 344.1 HU: Its converted CT value (CT divided by Au concentration) was as large as 8.0 × 103 HU/M. The r1 value of the Au/SiO2/GdC colloid solution was as large as 3.5 mM−1 s−1.

Gold (Au) is expected to function as a high contrast agent for X-ray computed tomography imaging as it has high X-ray absorptivity owing to a large atomic number compared to commercial materials such as barium and iodine. In addition, Au formation with a nanorod structure (AuNR) can be used for hyperthermia because of the enhancement of the photothermal reaction effect. The present work proposes a preparation method for core–shell nanoparticles composed of AuNR, SiO2, and a single nanometer-sized Au (sAu), toward imaging and photothermal therapy. The AuNRs (lateral size: 12.6 nm; longitudinal size: 44.9 nm) were silica-coated using a sol–gel method with tetraethyl orthosilicate, which produced AuNR/SiO2 nanoparticles with an average size of 86.9 nm. Thiol groups were introduced onto the AuNR/SiO2 nanoparticle surface by using (3-mercaptopropyl) trimethoxysilane (AuNR/SiO2-SH). The sAu nanoparticles (particle size: 2.6 nm) were produced by adding tetrakis (hydroxymethyl) phosphonium chloride as a reductant and HAuCl4 (aq) to NaOH solution. A thiol-terminated PEG (PEG-SH) aqueous solution and sAu nanoparticle colloidal solution were added in turn to the AuNR/SiO2-SH dispersed in EtOH (AuNR/SiO2/sAu-PEG). The CT value of the AuNR/SiO2/sAu-PEG particle solution was 702 HU. The CT values in tissues immediately increased after injection into mouse. Their blood retention times were 3–6 h, much longer than the few minutes of a commercial X-ray contrast agent (Iopamiron).

This study proposes a method for preparing high-concentration silica-coated Au (Au/SiO2) nanoparticles in colloidal solution from 1.5 × 10−3 M hydrogen tetrachloroaurate (III) trihydrate as the Au source and 1.0 × 10−2 M sodium citrate as the reducing reagent. The colloidal solution is applied to X-ray computed tomography (CT) imaging of mouse tissue. The Au nanoparticles in the colloidal solution had a diameter of 18.9 nm, and the Au concentration reached 1.5 × 10−3 M. The Au nanoparticles were silica-coated by modifying their surfaces with (3-aminopropyl)trimethoxysilane (APMS), then depositing silica nuclei generated by a sol–gel reaction of tetraethyl orthosilicate (TEOS) in water/ethanol initiated with sodium hydroxide (NaOH) on the surface modified with APMS. A colloidal solution of Au/SiO2 core–shell particles (silica shell thickness = 19.7 nm) was formed in a final as-prepared solution of 2.7 × 10−4 M Au, 2.0 × 10−5 M APMS, 24 M H2O, 1.9 × 10−3 M NaOH, and 4.1 × 10−3 M TEOS. The Au in the as-prepared colloidal solution was further concentrated to 0.27 M by salting-out and centrifugation. The CT value of the concentrated Au/SiO2 colloidal solution was 2.52 × 103 Hounsfield units, double that of a commercial X-ray contrast agent with the same I concentration as the Au concentration. When injected into mouse tissue, the Au/SiO2 colloidal solution demonstrated good imaging capability.

In tomato plants, Pepper mild mottle virus (PMMoV) cannot replicate because the tm-1 protein inhibits RNA replication. The resistance of tomato plants to PMMoV remains durable both in the field and under laboratory conditions. In this study, we constructed several mutant PMMoVs and analysed their abilities to replicate in tomato protoplasts and plants. We found that two mutants, PMMoV-899R,F976Y and PMMoV-899R,F976Y,D1098N, were able to replicate in tomato protoplasts, but only PMMoV-899R,F976Y,D1098N was able to multiply in tomato plants. Further analysis showed that the D1098N mutation of the replication proteins weakened the inhibitory effect of the tm-1 protein and enhanced the replication efficiency of PMMoV-899R,F976Y,D1098N. We also observed that the infectivity of the viruses decreased in the order wild-type PMMoV > PMMoV-899R,F976Y > PMMoV-899R,F976Y,D1098N in original host plants, pepper and tobacco plants. On the contrary, the single mutation D1098N abolished PMMoV replication in tobacco protoplasts. On the basis of these observations, it is likely that the deleterious side-effects of mutations in replication proteins prevent the emergence of PMMoV mutants that can overcome tm-1-mediated resistance.

Active hexose-correlated compound (AHCC) is an extract of a basidiomycete mushroom that enhances the therapeutic effects and reduces the side-effects of chemotherapy. Our previous studies demonstrated that heat-shock protein 27 (HSP27) was involved in gemcitabine-resistance of pancreatic cancer cells and it was down-regulated by AHCC-treatment. However, how AHCC down-regulated HSP27 is unknown. In the present study, we focused on two transcription factors reported to induce HSP27, heat shock factor 1 (HSF1) and high-mobility group box 1 (HMGB1) and investigated the effect of AHCC on their expression.KLM1-R cells were treated with AHCC and the protein expression of HSF1 and HMGB1 were analyzed by western blotting.The protein expression of HSF1 in KLM1-R was down-regulated by AHCC treatment. On the other hand, the protein expression of HMGB1 was not reduced in KLM1-R cells after AHCC treatment.The possibility that AHCC down-regulated HSP27 through down-regulation of the HSF1, was herein shown.

Results of our previous studies demonstrated that the expression of heat-shock protein 27 (HSP27) was increased and HSP27 was phosphorylated in the GEM-resistant pancreatic cancer cell line, KLM1-R. The expression of HSP27 is regulated mainly by heat-shock factor 1, but other transcription factors or kinases have been reported to activate HSP27. High-mobility group box 1 (HMGB1) is a nuclear transcription factor. It has been reported that HMGB1 regulates HSP27 gene expression. Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2) phosphorylates HSP27. In the present study, we investigated the expression of HMGB1 and MAPKAPK2 in KLM1-R cells.The expression levels of HMGB1 and MAPKAPK2 were compared between KLM1 and KLM1-R cells by western blotting.The protein expression of both HMGB1 and MAPKAPK2 were increased in KLM1-R cells compared to KLM1 cells.The increase of both HMGB1 and MAPKAPK2 in KLM1-R cells compared to KLM1 suggest the possibility of the activation of the pathway of HSP27 by HMGB1 and MAPKAPK2 in gemcitabine-resistant KLM1-R cells.

In tomato plants, Pepper mild mottle virus (PMMoV) cannot replicate because the tm‐1 protein inhibits RNA replication. The resistance of tomato plants to PMMoV remains durable both in the field and under laboratory conditions. In this study, we constructed several mutant PMMoVs and analysed their abilities to replicate in tomato protoplasts and plants. We found that two mutants, PMMoV‐899R,F976Y and PMMoV‐899R,F976Y,D1098N, were able to replicate in tomato protoplasts, but only PMMoV‐899R,F976Y,D1098N was able to multiply in tomato plants. Further analysis showed that the D1098N mutation of the replication proteins weakened the inhibitory effect of the tm‐1 protein and enhanced the replication efficiency of PMMoV‐899R,F976Y,D1098N. We also observed that the infectivity of the viruses decreased in the order wild‐type PMMoV > PMMoV‐899R,F976Y > PMMoV‐899R,F976Y,D1098N in original host plants, pepper and tobacco plants. On the contrary, the single mutation D1098N abolished PMMoV replication in tobacco protoplasts. On the basis of these observations, it is likely that the deleterious side‐effects of mutations in replication proteins prevent the emergence of PMMoV mutants that can overcome tm‐1‐mediated resistance.

Code clone is a code fragment that has identical or similar fragments to it in the source code. Fowler and Kereivsky wrote several techniques to remove code clones in refactoring patterns that they documented. Those refactoring patterns include characteristics of code clone and corresponding steps to merge code clones. However, according to our experience in code clone research field, a lot of different types exist between code clones similar to each others, and most of those difference types are not mentioned in previous refactoring pattern. It is useful to develop a collection of patterns for clone refactoring based on precise code clone categorization. In this paper, we describe an approach to developing our intended collection of refactoring patterns and then propose an example of refactoring pattern that we have developed.

Introduction Chemokines are regulated by a family of ‘atypical’ chemokine receptors, D6, DARC and CCX-CKR, each of which efficiently internalizes its cognate chemokine ligands. Development of monoclonal antibodies (MAbs) that would recognize CCX-CKR on the cell surface will be helpful to identify primary CCX-CKR-expressing cell types and analyze the fate of CCX-CKR after ligand binding to the receptor. Methods We generated IgG MAbs recognizing the cell-surface CCX-CKR by DNA immunization using a molecular adjuvant, and analyzed the epitope recognized by the MAbs. Then, the reactivities of the MAbs with CCX-CKR-transfected cells, and also hepatocytes and hepatic tumor lines were evaluated. Finally, we also tested the ligand-like activities of the MAbs, namely, induction of internalization of CCX-CKR by the MAbs. Results A panel of MAbs reacting with CCX-CKR expressed on the cell surface was prepared. The panel was a small one, consisting of only ten MAbs, but was rich in terms of diversity of the Ig isotypes and of the epitopes. Epitope analyses revealed that all the 10 MAbs recognized at least three different, although very close, peptide structures of the N-terminal domain. Three MAbs, namely, 2F11, 13E11 and 14F10, were selected to represent the panel. All of the MAbs were applicable for flow cytometry and immunoflurescent assays and immunoprecipitation. The reactivity of the 2F11 MAb was also confirmed by western blotting. Endogenous expression of CCX-CKR on human hepatocytes and hepatic tumor cell lines was demonstrated using the 13E11 MAb. Interestingly, binding of the 13E11 MAb with B300-19 cells expressing CCX-CKR resulted in induction of CCX-CKR internalization. Discussion This panel of MAbs may be expected to prove valuable for further study of the functions of this silent chemokine receptor, including those related to the homeostasis of lymphoid cells, and to the growth and metastasis of hepatic cancer.

Nitrogen-doped ZnO nanoparticles were prepared by a gas evaporation method. We chose a drop coating method for the deposition of thin films from a dispersion of the nanoparticles in water. The drop coating method appears to be the optimum method for the deposition of thin films from the dispersions. Sintering at 300 °C improved the deposition of a thin film compared with that of films dried at room temperature. Photoluminescence spectroscopy and scanning electron microscopy showed that high-density aggregated films were formed by the drop coating method.

Urinary neutral oligosaccharides of various connective tissue diseases were studied by gel-filtration through Sephadex G--10 after treatments with cetylpyridinium chloride (CPC), Dowex 50 (H+ form) and Dowex 1 (Cl- form), in succession. Increased excretion of urinary glucose-containing oligosaccharides, specifically glucosylgalactose was observed in most of the patients with chondrosarcoma, rheumatoid arthritis, Werner's syndrome, Rothmund Thomson syndrome and Morquio's disease. However, urinary excretion of neutral oligosaccharides in the patients with osteosarcoma and other tumorous conditions, and some systemic disorders in the connective tissues, examined in the present study, showed almost normal values. It is indicated, therefore, that the activity of glucosidase in insufficient for the glucose-containing oligosaccharides produced from the ground substance(s) in the former type connective tissue diseases.

A classic case of progeria (Hutchinson-Gilford syndrome) in a 9-year-old Japanese boy is presented. The characteristic clinical features in this patient were similar to those reported in the literature. The total amount of acid glycosaminoglycans excreted in the urine was within the normal range, but there was an increase in hyaluronic acid excretion. The hyaluronuria was a novel finding in progeria, providing a common linkage with the hyaluronuria found in Werner's syndrome.

Normal human urine was treated with cetylpyridinium chloride (CPC), and the CPC-supernatant was passed through columns of Dowex 50 X 2 (H+ form) and Dowex 1 X 2(Cl-form) in succession. The concentrated effluent containing neutral saccharides was then gel-filtered through a column of Sephadex G-10. The resulting effluent was divided into 5 subfractions (SFs. 1-5). Comparison of hexose contents of the subfractions indicated that healthy individuals regularly excreted neutral oligosaccharides in urine with a similar pattern regardless of age or sex. Component A isolated from SF2 was characterized as glucosylgalactose by paper chromatography before and after acid hydrolysis of the intact one and its reduction product.

TOKUNAGA, M., WAKAMATSU, E., SATO, M., NAMIKI, O., YOKOSAWA, A. and MOTOMIYA, M. Lipid Composition of Adipose Tissue from “Membranous Lipodystrophy”. Tohoku J. exp. Med., 1981, 133 (4), 451-456 -The lipid composition of the adipose tissue from the bone marrow and that of the subcutaneous depot fat of four patients of membranous lipodystrophy were investigated. It was found that the lipid composition of the samples from the cases of membranous lipodystrophy was almost similar to that in controls. Also discussions were made on mechanism related to the accumulation of abnormal depot fat.

Although excretion of acid glycosaminoglycans into urine of five patients with Werner's syndrome were within normal limits, the quantity of hyaluronic acid increased in this disease. To this novel finding, a term “Hyaluronuria” was proposed.

The exercretion of urinary total glycosaminoglycans (GAG) in a case of Rothmund Thomson syndrome associated with osteosarcoma was increased about 2--3 times that of normal control. Since the excretion of urinary total GAG in two cases of osteosarcoma was within normal range, the increased excretion of total GAG might be on account of the metabolic disorder of GAG in Rothmund Thomson syndrome. The prominent fractions obtained by Dowex 1 column chromatography from this syndrome were 0.75 M Fr and 1.0 M Fr, in which the major GAG were indicated to be partially degraded forms of heparan sulfate and chondroitin sulfate, respectively.