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Hideaki Okawada, Yuta Saito, Shigehiro Iwabuchi, Shotaro Kosuge, Haruo Takahashi, Hidetoshi Onda, Masahiro Kito Department of Ophthalmology, Showa University, Tokyo, Japan Purpose: Vascular endothelial growth factor (VEGF) plays an important role in branch retinal vein occlusion (BRVO) with cystoid macular edema (CME). Monitoring changes in VEGF is crucial for evaluating treatment but requires vitreous or aqueous humor sampling, which hampers its clinical application. We investigated the correlation between VEGF and protein concentration in the aqueous humor (flare) and whether this could be used to monitor treatment-related VEGF changes. Design: This retrospective observational study involved 19 previously untreated patients with BRVO. Aqueous humor was obtained, and intravitreal ranibizumab (IVR) injection was administered to these patients. The correlation between VEGF and flare, central retinal thickness (CRT), and best-corrected visual acuity (BCVA) was investigated. Differences in these values were considered between pre-IVR and 1week and 1–3months post-IVR. Moreover, in patients with recurrence who received additional IVR, further changes in VEGF were examined. Main outcome measures: The end point of this study was BCVA, flare, and CRT at the fovea. Results: Significant improvement was seen in BCVA and CRT at all time points and in Flare at 1 vs 3months post-IVR; nevertheless, additional IVR was necessary in 94% of cases. In a patient with recurrence, CRT did not improve, even though VEGF decreased. Conclusion: Flare may be effective for estimating VEGF levels in aqueous humor pre-IVR. Inflammation-related molecules other than VEGF may be related to recurrence. Keywords: aqueous humor, branch retinal vein occlusion, flare, protein density, ranibizumab, vascular endothelial growth factor

Hepatocyte growth factor (HGF) activator is a serine protease that is produced and secreted by the liver and circulates in the blood as an inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF activator from the conditioned medium of a human MKN45 stomach carcinoma cell line and molecularly cloned its cDNA. The sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, suggesting that the inhibitor is a member of the Kunitz family of serine protease inhibitors. The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence at the COOH-terminal region. Inhibitory activity toward HGF activator was detected in the membrane fraction as well as in the conditioned medium of MKN45 cells. These results suggest that the inhibitor may be produced as a membrane-associated form and secreted by the producing cells as a proteolytically truncated form.

Clarification of the degradation mechanism of photoluminescence (PL) charac-teristics of InGaAsP compressively strained layer multi-quantum well (SL-MQW) structures on an InP substrate is studied to realize highly strained SL-MQW lasers with a large number of well layers. An unusual temperature-dependence of the PL peak wavelength is observed in the SL-MQW structure: the shift of the PL peak wavelength is reduced with change in measurement temperature. The degradation of PL characteristics is evaluated by the reduc-tion in the PL shift as well as an increase in PL linewidth and a decrease in PL peak intensity. The extent of the PL degradation increases with an increase in the number of well layers and an increase in strain. In the SL-MQW structure with degraded PL characteristics, the structure of CuPt-type atomic ordering is clearly observed for the first time in each barrier layer by high-resolution transmission electron microscope (HR-TEM) measurements. In the SL-MQW structure, neither dislocation nor three-dimensional nucleation is observed by TEM measurements. By increasing the growth temperature, elimination of the ordering structure as well as improvement in the PL characteristics have been confirmed even in the SL-MQW structure with a large amount of strain and a large number of well layers.

We have previously shown that the Saccharomyces cerevisiae USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a 200-kDa protein (1,790 amino acids) which is present in a nonglobular high molecular mass complex. Antibodies against an N-terminal portion of Uso1 protein recognized a 100-kDa protein in Western blot of the temperature-sensitive uso1-1 mutant cell lysate. The nucleotide sequence of uso1-1 indicated the 951st codon was UAG (amber) in place of CAG (glutamine) in USO1. Deletion study of USO1 gene indicated that such truncated Uso1 polypeptides are sufficiently functional at 25 degrees C but not at 37 degrees C. Mutant Uso1-1 protein displayed an apparent molecular mass of 400-500 kDa in gel filtration while it cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa), in sucrose density gradient centrifugation. These results indicated that truncated Uso1-1 protein is still present in a nonglobular high molecular mass complex, similar to the wild-type Uso1 protein.

Copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn) levels in organs of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis, were determined by instrumental neutron activation analysis method. Unusual accumulations of Cu in the liver of LEC rats were found, depending on the age of the animals, the metal concentration being more than approximately 20-40 times those of normal LEA rats (Long-Evans rats with an agouti coat color). Fe and Zn were also accumulated, in addition to Cu, significantly in the LEC rats. The unusual Cu accumulations in the liver of LEC rats were associated with the induction of metallothionein, estimated by radioimmunoassay method, in the liver of LEC rats, rather than that of superoxide dismutase, estimated by electron spin resonance -spin trapping method. These findings suggest that the unusual Cu accumulation in LEC rats is involved in the development of jaundice, hepatic injury and hepatocellular carcinoma.

A system is described that identifies a number of unknown drugs in blood specimens for clinical-toxicological purposes. Reversed-phase (C 18 ) high-performance liquid chromatography with a photodiode array detector saves the ultraviolet (UV) spectrum of every chromatographically significant peak. The relative retention times could be used as one element of the identification; the second element is the UV spectrum, which is recorded on-line during the separation. The development of a computer program enabled complementary color points to be calculated from the UV spectrum. An evaluation of these color points for identifying the drugs has been made. Precisions of approximately 2% relative standard deviation can be achieved for complementary color points obtained from 18 different drugs. The combination of Chromatographic and complementary color points selects the most suitable drug, which could be the unknown.

GaN films having distinct p-type conductivity, with hole concentration up to 4 × 10 16 cm 3 at RT, have been achieved for the first time. Samples were grown by MOVPE on a sapphire substrate using the AIN buffer layer. LEEBI treatment is found to be effective to lower the resistivity of the GaN:Mg, and to get the p-type GaN. The p-n junction LED, which emits UV light and violet-blue light at RT, is fabricated for the first time.

We achieved successful growth of a high quality undoped GaN film, in which photopumped stimulated emission at RT were observed for the first time. Different configuration measurements showed that stimulated emission occurred at about 83 meV lower than the peak photon energy of spontaneous emission, at RT.

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C 18 column (15 cm × 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 m M sodium 1-hexanesulphonate and 0.1 m M p -hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p -hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30–200 pg per injection.

7-(Diethylamino)-3-[4-((iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA) and 4-(bromomethyl)-7-methoxycoumarin have been evaluated as fluoropyrimidine-derivatizing agents to be detected using peroxyoxalate chemiluminescence with high-performance liquid chromatography. The derivatization procedure required only one step. No chemiluminescence was observed from the bromo derivatives, and the detection limits of fluoropyrimidine compounds derivatized with the iodo compound and detected with peroxyoxalate chemiluminescence were in the low femtomole range.

高感度な化学発光HPLCでピリミジン化合物を定量するために蛍光標識体となるべきクマリン誘導体を検索したところ7-diethylamino-3-[4-((iodoacetyl)amino)phenyl]-4-methylcoumarine(DCIA)が適していた.このためDCIAによるピリミジン化合物のプレラベル化を行い,クロマトグラフィー特性を調べると共に測定感度を上昇させることを試みた.DCIAは無水炭酸カリウムと18-crown-6の共存下アセトン中で目的物と反応させた.DCIA誘導体の分離は逆相クロマトグラフィーで行い検出には化学発光を用いた.化学発光はDCIA-Bis(2,4,6-trichlorophenyl)oxalate-過酸化水素系により発生させた.反応条件,クロマトグラフィー条件及び測定系などの最適化を行った結果,誘導体化された試料の検出限界は注入量で5-fluorouracilは19 fmol, uracilは22.5 fmol及び5-fluorouridineは10 fmolであった.又,これらの薬物が血清1 ml当たり1.25～250ng含まれる濃度範囲で検量線の直線関係が得られた.

A method for the chemiluminescent detection of fluoropyrimidine compounds with 7-(diethylamino)-3- {4[(iodoacetyl)amino]phenyl}-4-methylcoumarin using peroxyoxalate is described. The procedure is rapid, simple and requires little or no experience in labelling techniques. The amounts of the derivatives are linearly related to the amount of the starting fluoropyrimidine compounds and the procedure can therefore be used in the determination of these solutes. Using reversed-phase liquid chromatography, detection levels of 20–40 fmol could be realized.

High output-power and narrow spectral-linewidth operation of 1.3 /spl mu/m gain-coupled distributed feedback (DFB) lasers with InAsP buried absorptive grating are reported. In this report, we have investigated the effect of the thickness of separate-confinement-heterostructure (SCH) layer on the spectral linewidth. As a result, it was clarified that decreasing the thickness of SCH layer is effective to reduce the spectral linewidth. For the fabricated laser, a record high output-power of 230 mW and a minimum spectral-linewidth of 100 kHz was realized.

Characteristics of the InAsP layer formed on a corrugated InP substrate by a mass-transport mechanism have been investigated for use as the buried grating of distributed feedback (DFB) lasers, The size of the InAsP layer can be controlled by the height of the corrugation, and the arsenic composition in the InAsP layer can be controlled by the AsH/sub 3/ partial pressure. The results of TEM, EDS and PL show that InP is suitable as the buffer layer between the InAsP layer and MQW active layer. Fabricated 1.3 /spl mu/m DFB lasers which have an InAsP layer as an absorptive grating have shown low threshold current and high slope efficiency from -40-+85/spl deg/C, and high reliability has been demonstrated.

The dependence of photoluminescence (PL) characteristics on both SL-MQW structure and epitaxial growth condition is studied. The degradation of PL characteristics in the SL-MQW is confirmed as a broadening of PL linewidth and an unusual temperature-dependence of PL peak wavelength. By using a high-resolution transmission electron microscope (HRTEM), the structure of atomic ordering is demonstrated over entire barrier layers of the SL-MQW structure with degraded PL characteristics. By increasing growth temperature, elimination of the ordering structure as well as improvement of the PL characteristics have been confirmed even in a SL-MQW structure a with large amount of strain.

Saccharomyces cerevisiae uso1-1 mutant stops the transport of secretory proteins from the endoplasmic reticulum to the Golgi apparatus at 37 degrees C. We found that this temperature-sensitive defect was suppressed either by increasing the concentration of calcium ion in the medium or by introducing in the cell the SLY genes which suppress the defect of Ypt1 protein, a small GTP-binding protein. The common phenotype and suppression of the mutants suggest that Uso1 and Ypt1 proteins function in the same process of protein transport, i.e., targeting or fusion of the transport vesicles to the Golgi membrane.

We have previously shown that USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a hydrophilic protein of 1790 amino acids. The sequence of carboxyl-terminal 1010 amino acids was predicted to have an alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. Antibodies raised against partial sequences of the Uso1 polypeptide reacted with a 200 kDa protein in Western blots of the wild-type yeast proteins. The Uso1 protein was found predominantly in the soluble fraction and displayed a molecular mass of 800-900 kDa in gel filtration when globular protein were used as molecular mass standards. In sucrose density gradient centrifugation, however, the Uso1 protein cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa). These results suggest that, in its native state, the Uso1 protein forms a nonglobular oligomer.

Recently, copper(Cu) was found to be unusually accumulated, suggesting the induction of metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis. Thus, the direct relationship between the unusual Cu accumulation and the induction of Cu-MT was investigated by giving LEC rats Cu-overloaded or Cu-deficient diets. Results based on the determinations of Cu and MT levels in several organs, as well as the gel-filtration profiles of the cytosols of liver homogenates, showed that dietary Cu induced Cu-MT and development of hepatic injury associated with jaundice.

High output power and narrow spectral linewidth operation of 1.3 µm gain-coupled distributed feedback (DFB) lasers with InAsP buried absorptive grating is reported. In this study, we have investigated the effect of the thickness of a separate-confinement-heterostructure (SCH) layer on the single-mode stability and the spectral-linewidth property of 1.3 µm gain-coupled DFB lasers. As a result, we clarified that decreasing the thickness of the SCH layer led to stable single-mode operation at high output power and the reduction of the product of the spectral linewidth and the output power. For the fabricated gain-coupled DFB laser, a record high output power of 230 mW and a minimum spectral linewidth of 100 kHz at an output power of 150 mW were realized.

A new structure of 1.3 µm strained-layer multi-quantum well (MQW) complex-coupled distributed feedback lasers having an InAsP absorptive grating is demonstrated. In this structure, the InAsP absorptive grating and the strained-layer MQW active layer can be fabricated by a metalorganic vapor phase epitaxy process involving only one step. Furthermore, the fabricated laser has a low threshold current (16.8 mA) and a high efficiency (0.45 mW/mA).

A selective and sensitive assay of substrates (hypoxanthine, xanthine and allopurinol) of xanthine oxidase by reversed-phase liquid chromatography coupled with the use of immobilized enzyme reactors is described. These compounds were oxidized by immobilized xanthine oxidase and produced hydrogen peroxide, which was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. The detection limits of hypoxanthine, xanthine and allopurinol were approximately 50, 120 and 130 pg per injection, respectively.Immobilized xanthine oxidase inhibited by oxipurinol during the assay was reactivated by 2, 6-dichlorophenolin-dophenol and could be used for a long period without a significant activity loss. These methods were applied to plasma and urine samples.

A selective and sensitive assay of hypoxanthine and xanthine in biological fluids by high-performance liquid chromatography coupled with immobilized-enzyme reactors was developed. The separations were achieved by reversed-phase liquid chromatography. Hydrogen peroxide produced from hypoxanthine and xanthine by immobilized xanthine oxidase was determined fluorometrically using immobilized peroxidase and p -hydroxyphenylacetic acid. Immobilized enzymes were prepared by intermolecular cross-linking to controlled-pore glass. Assay of allopurinol was also possible by the present method. The method was applied to scrum and urine, the detection limits of hypoxanthine and xanthine were approximately 50 and 120 pg per injection, respectively.

A Selective and sensitive assay of hypoxyanthine, xanthine and inosine by reversed-phase liquid chromatography coupled with immobilized enzyme reactors is described. The flourometric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of hypoxanthine, xanthine and inosine, which were oxidized to hydrogen peroxide in the presence of the immobilized enzymes (purine nucleoside phosphorylase and/or xanthine oxidase. The enzymes were immobilized the the intermolecular cross-linking method on controlled pore-glass. The method established was applied to serum and urine samples. The detection limits of hypoxanthine, xanthine and inosine were approximately 130, 300 and 650 pg per injection, respectively.

Immobilized xanthine oxidase was prepared by intermolecular cross-linking to porous aminopropyl-glass beads, and the properties were investigated in a batch process. Simultaneous analysis of hypoxanthine, xanthine and uric acid was achieved by using small column packed with the immobilized xanthine oxidase as a reactor for high-performance liquid chromatography (HPLC).

A thin AlN buffer layer was proven to be effective on both sapphire A-face and sapphire C-face substrates for the preparation of high quality Zn-doped as well as undoped GaN. The nature of Zn-related luminescence centers and their density are strongly affected by growth conditions; especially by the substrate temperature, which must be lower than 950°C for the efficient formation of blue luminescence centers. Zn-doped GaN grown on a sapphire A-face was found to have a greater density of blue luminescence centers than that on a C-face, indicating that the A-face is superior to the C-face as a substrate.

The aromatic methyl group migration and hydroxylation of p-toluidine by several Fe (II)-thiol compound and hemin-thiol compound systems were investigated as a model of cytochrome P-450 of liver microsomes. In these systems, 4-hydroxy-m-toluidine, 3-hydroxy-p-toluidine and 2-hydroxy-p-toluidine were formed as the main products of the reaction. The yield of methyl group migration and hydroxylation were dependent on the type of thiol compounds, chemical form of iron (Fe (II) or hemin), pH of the reaction mixture, reaction periods as well as on the concentration of the substrates or the thiol compounds. These results on the methyl group migration of p-toluidine suggest that the Fe (II)-thiol and hemin-thiol systems can be regarded as a good chemical model for the study of the function and structure of cytochrome P-450.

Distinct p-type conduction is realized with Mg-doped GaN by the low-energy electron-beam irradiation (LEEBI) treatment, and the properties of the GaN p-n junction LED are reported for the first time. It was found that the LEEBI treatment drastically lowers the resistivity and remarkably enhances the PL efficiency of MOVPE-grown Mg-doped GaN. The Hall effect measurement of this Mg-doped GaN treated with LEEBI at room temperature showed that the hole concentration is ∼2·1016cm-3, the hole mobility is ∼8 cm2/V·s and the resistivity is ∼35 Ω·cm. The p-n junction LED using Mg-doped GaN treated with LEEBI as the p-type material showed strong near-band-edge emission due to the hole injection from the p-layer to the n-layer at room temperature.