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Based on a two-dimensional percolation theory and an autocatalytic rate expression for oil autoxidation, a statistical model is proposed to estimate the effects of the oil droplet size and the volume fraction of oil within the microcapsule on the oxidation of microencapsulated oil, by means of computational simulations. The model can address both the unique oxidation steps for microencapsulated oil: The rapid progress of oxidation at the early stage of storage and the leveling off of the oxidation after prolonged storage. The simulation also demonstrates that the reduction of the oil droplet size retards the oxidation of microencapsulated oil as well as lowering of the oil fraction within the microcapsule. Practical Applications: A statistical model is proposed to simulate oxidation process of microencapsulated oil. The simulation enable us to reasonably design oil microencapsulation. Based on a two-dimensional percolation theory and an autocatalytic rate expression for oil autoxidation, a statistical model is proposed to estimate the effects of the oil droplet size and the volume fraction of oil within the microcapsule on the oxidation of microencapsulated oil, by means of computational simulations.

We report a case of unilateral blindness due to ophthalmic artery occlusion following injection of hyaluronic acid into the glabellar area for facial soft-tissue augmentation.A 20-year old woman underwent injection of hyaluronic acid into the glabellar area at an aesthetic plastic clinic. Immediately after injection, she suffered nausea, pain, paralysis of limbs and visual loss in her right eye. Hyaluronidase was instanly injected into the same place, but these symptoms did not improve. She was transferred to our hospital's emergency department. At the first examination, she had no light perception in the right eye and her right pupil was dilated. The fundus examination revealed right central artery occlusion. No significant findings were detected in her head. Eye ball massage, hyperbaric oxygen therapy, and drip infusion of urokinase were conducted, but no improvement resulted. Fluorescein and indocyanine green angiography showed no filling of the right retinal and choroidal arteries. Electroretinogram was completely disappeared. Magnetic resonance angiography could not reveal the obstruction point of the ophthalmic artery.Ophthalmic artery occlusion was believed to have occured after injection of hyaluronic acid into the glabellar area.

Purpose To determine the effect of high doses of AGEs on neurite regeneration in isolated rat retinas, and the regenerative effects of different neurotrophic factors. Methods Retinal explants of 4 adult SD rats were three-dimensionally cultured in collagen gel, and incubated in: 1) serum free control culture media; 2) 100 μg/ml glucose-AGE-BSA, glycolaldehyde-AGE-BSA, glyceraldehyde-AGE-BSA media; 3) glucose, glycol, glycer+100 ng/ml neurotrophin 4 (NT-4)media; 4) glucose, glycol, glycer+100 ng/ml hepatocyte growth factor (HGF) media; 5)glucose, glycol, glycer+100 ng/ml glial cell line derived neurotrophic factor (GDNF) media; or 6) glucose, glycol, glycer+100 ng/ml tauroursodeoxycholic acid (TUDCA) media. After 7 days, the number of regenerating neurites were counted under a phase-contrast microscope. The explants were immunostained for NFkB transcription factor. Statistical analyses were performed by one-way ANOVA. Results In retinas incubated with AGEs, the number of regenerating neurites were fewer than in control. Neurotrophic factors increased the number of neurites, but more significantly in the NT4 group. The number of NFkB immunopositive cells was higher in retinas exposed to AGEs than in control (53,2 ± 7,2% vs.31,6 ± 16,08%,P = 0,0146). Neurotrophic factors decreased the number of immunopositives cell, but more significantly in the NT4 group. Conclusion High dose AGEs induce activation of NFkB and NT4 significantly suppresses this activation and enhances neurite regeneration in isolated retinas compared with other neurotrophic factors such as HGF,GDNF,and TUDCA.

Purpose We present a case of orbital mucosa-associated lymphoid tissue lymphoma accompanied with refractory serous retinal detachment. Methods A 44-year-old man who developed uveitis in the right eye was referred to Chiba National Hospital. His visual acuity was 0.5, and the IOP was 34 mmHg OD. Keratic precipitates and inflammatory cells were observed in the anterior chamber. Fundus examinations showed a serous retinal detachment (SRD) and a hyperemic disc. B-mode ultrasonography showed a thickened choroidal membrane and a retrobulbar mass. The MRI findings showed that the mass had T1- and T2-low intensities. Although topical steroid treatments improved the inflammation, the SRD was not improved. Because the oral steroid administration did not reduce the size of the retrobulbar mass, he was referred to the Chiba University Hospital. Results Two month later, the retrobulbar mass had increased, and a partial resection biopsy combined with flow cytometry was performed. The final diagnosis was mucosa-associated lymphoid tissue lymphoma. The intraocular IL-10 was not measurable. After R-CHOP chemotherapy, the tumor size decreased but the SRD was not improved. Not only lymphoma cell infiltration but also choroidal circulatory disturbance may be associated with the development of the SRD. Conclusion Retrobulbar malignant lymphomas accompanied by refractory SRD are very rare and difficult to diagnose without biopsy. But, early diagnosis is more helpful in deciding the appropriate therapy.

The study was conducted to determine whether serum vascular endothelial growth factor (VEGF) levels are significantly correlated with subfoveal choroidal thickness (CT) and foveal thickness (FT) in patients with polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome.In this cross-sectional observational case series, we studied 31 eyes of 16 treatment-naïve patients with POEMS syndrome with no evidence of fundus abnormalities. Subfoveal CT and FT were measured using enhanced depth imaging optical coherence tomography (EDI-OCT), and correlations between serum VEGF levels and subfoveal CT and FT were determined.The mean subfoveal CT was 417.9 ± 73.5 μm (right eye, 416.7 ± 81.2 μm; left eye, 419.0 ± 68.1 μm), and the mean FT was 243.8 ± 35.2 μm (right eye, 248.8 ± 22.0 μm; left eye, 239.1 ± 44.6 μm). There was a significant positive correlation between the serum VEGF level and subfoveal CT (right eye, r = 0.58, p = 0.021; left eye, r = 0.60, p = 0.012), but the correlation between the level of serum VEGF and FT was not significant (right eye, r = 0.007, p 0.05; left eye, r = 0.25, p 0.05).The significant correlation between the serum VEGF level and subfoveal CT in patients with POEMS syndrome suggests that choroidal thickness is influenced by the level of serum VEGF. These results not only aid in an understanding of the pathogenesis of ocular changes in patients with POEMS syndrome, but also offer clues regarding the pathogenesis of other choroidal diseases.

Alpha-B crystallin (αB-crystallin) is a molecular chaperone, and one of the small heat shock proteins (sHSPs). αB-crystallin has been shown to have neuroprotective properties. The purpose of this study was to determine the level of αB-crystallin in eyes with a rhegmatogenous retinal detachment (RRD). Vitreous samples were collected before vitrectomy from 32 eyes of 32 consecutive patients with RRD. The concentration of αB-crystallin was measured by enzyme-linked immunosorbent assay (ELISA). Vitreous samples were also collected before vitrectomy from 16 patients with an idiopathic macular hole and epiretinal membrane, and used as controls. The mean age was 56 years (range, 31–67 years) in the RRD cases and 66 years (range, 49–77 years) in the controls. The mean level of αB-crystallin was significantly higher in the RRD cases (36.7 ± 37.4 ng/ml) than that in controls (8.5 ± 12.2 ng/ml; P = 0.0028). The increased level of αB-crystallin suggests that αB-crystallin may play a neuroprotective role by lessening retinal cell death after a retinal detachment.

Recent studies have shown that the glycation-associated damage is not limited to patients with diabetes. Because of their association with oxidative stress, advanced glycation end-products (AGEs) have also been implicated in many neurodegenerative diseases, such as Huntington disease, amyotrophic lateral sclerosis, and Alzheimer disease (Yan et al., 1996; Ma and Nicholson, 2004). As in other age-dependent neurodegenerative diseases of the brain, oxidative stress-associated age-dependent pathogenic processes are not unexpected in glaucoma because this disease is also more common in the elderly (Tezel et al. 2007). Growing evidence indicates that neuronal abnormalities including neuronal cell death are associated with the pathogenesis of early diabetic retinopathy. Our previous studies of human retinas indicate that mitochondrial- and caspase-dependent cell death pathways are associated with retinal neuronal cell degeneration in patients with diabetes (Oshitari et al., 2008). Brain-derived neurotrophic factor, neurotrophin-4 (NT-4), vascular endothelial growth factor (VEGF)120, VEGF164, taurine-conjugated ursodeoxycholic acid and citicoline had been studied and showed a survival effect on damaged retinal neurons induced by diabetic stress (Oshitari et al. 2003, 2010). It was also found that NT-4 had the best neuroprotective and regenerative effect under high glucose conditions (Oshitari et al, 2010). Earlier study indicated that the maximum rescue ratio of caspase-1, -3, -8, and -9 inhibitors in cultured retinas was 60% in damaged retinal ganglion cells (RGCs) (Oshitari and Adachi-Usami, 2003). Thus, at least 40% of neuronal cell death in damaged RGCs in cultured retinas should be related to caspase-independent cell death mechanisms. However, no reports have focused on caspase-independent cell death pathways under diabetic stress including AGEs exposure. Apoptosis-inducing factor (AIF) was the first mitochondrial protein shown to mediate cell death independent of caspase (Susin et al., 1996, 1999). It was initially characterized as a mitochondrial protein confined in the intermembrane space of healthy cells. In healthy cells, AIF is a mitochondrial flavin adenine dinucleotide (FAD)-dependent oxidoreductase that plays roles in redox control and oxidative phosphorylation (Modjtahedi et al., 2006).

Masayasu Kitahashi,1 Takayuki Baba,1 Madoka Sakurai,1 Hirotaka Yokouchi,1 Mariko Kubota-Taniai,1 Yoshinori Mitamura,2 Shuichi Yamamoto1 1Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba, 2Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan Background: The purpose of this study was to compare the effectiveness of pneumatic displacement combined with intravitreal bevacizumab (IVB) with that of pneumatic displacement (PD) alone to treat massive submacular hemorrhage (SMH) secondary to polypoidal choroidal vasculopathy (PCV). Methods: Thirty-two eyes of 32 patients with massive SMH secondary to PCV were studied. Twenty-two eyes were treated with a combination of PD and 1.25 mg of intravitreal bevacizumab (PD + IVB group), and ten eyes with pneumatic displacement alone (PD group). Results: Pretreatment, the differences in best-corrected visual acuity and size of the SMH between the two groups were not significant (P=0.59 and P=0.72, respectively). Complete displacement of the hemorrhage from under the fovea was achieved in 19 of 22 eyes (86.4%) in the PD + IVB group and in five of ten eyes (50%) in the PD group. The best-corrected visual acuity in the PD + IVB group was significantly better than that in the PD group at one, 3, and 6 months after treatment (P0.3 logMAR units was obtained in 18 eyes (81.8%) in the PD + IVB group and two eyes (20%) in the PD group (P

A simple method for determining concentration (water or solvent content) dependent diffusivity in liquid foods and polymer solutions from the regular regime drying curve was developed on the basis of the power-law diffusion model. The method was first tested to the drying data generated from numerical solutions of the diffusion equation with various types of concentration dependent diffusivities. The present method was found to be very simple yet accurate compared with the previous methods. Then, the drying rates of various sugar solutions experimentally obtained were analyzed on the basis of the present method to determine the moisture content dependent diffusivities.

This study examined the interaction mechanisms of beta-lactoglobulins A and B (Lg A, Lg B) with an anion exchanger, Q-Sepharose at pH near the isoelectric point at which the proteins are expected to be electrically neutralized under various NaCl concentrations and temperatures by the equilibrium binding analysis and the adsorption enthalpy directly measured by isothermal titration calorimetry. The data evaluated from isotherms fitted by the Langmuirean model reveal that the addition of NaCl considerably reduced the binding affinities and capacities of both the proteins with Q-Sepharose at pH 5.2, indicating that electrostatic forces are dominant during the adsorption. However, the hydrophobic interaction seems to be involved in adsorption as well at a higher NaCl concentration, and the adsorption enthalpies confirm this suggestion. In addition, the effects of temperature on the equilibrium binding behaviors for Lg A or Lg B with Q-Sepharose were found to be salt concentration-dependent, probably due to their different binding mechanisms at 0.03 M and 0.3 M NaCl. Where, at 0.3 M NaCl, the hydrophobic interaction plays a more pronounced role. This implication was again supported by the adsorption enthalpies. The presented data provide further insight to the interaction mechanisms between proteins and ion exchangers, facilitating the optimization of protein separations.

Purpose: To determine the correlation between the tomographic features and the visual functions of eyes with diabetic macular edema. Methods: Optical coherence tomographic (OCT) images and multifocal electroretinograms were obtained from 45 eyes of 25 patients with diabetic macular edema and from 21 eyes of 21 normal subjects. Results: The OCT images showed cystoid macular edema in 18 eyes and diffuse retinal swelling without cystoid edema in the other 27 eyes. The fovea was significantly thicker in eyes with cystoid macular edema and in those with diffuse retinal swelling than in normal eyes. The fovea of eyes with cystoid edema was significantly thicker than the fovea of eyes with diffuse swelling. The best-corrected visual acuity and the electrical response density from the macular area were significantly reduced in eyes with diabetic macular edema, particularly in those with cystoid edema. The best-corrected visual acuity and macular response density of the multifocal ERGs were inversely correlated, and the implicit times were directly correlated with foveal thickness. Conclusion: The visual acuity was correlated significantly with morphological changes revealed by OCT and with multifocal ERGs. The combination of OCT and multifocal ERGs may provide objective criteria for the evaluation and assessment of diabetic macular edema.

ヒドロキシアパタイトクロマトグラフィー（HAC）とイオン交換クロマトグラフィー（IEC）の分子認識機構をIECの工学的解析のために考案した数学モデルを適用することにより解析した．β-lactoglobulin（Lg）とRibonucleaseA（RNaseA）をモデル試料とし，勾配溶出実験をさまざまな移動相pHについて行い溶出塩濃度（1，）並びに吸着サイト数（B）を決定した．IECではLg，RNaseAともにpHが等電点（pl）から離れるに従ってIR並びにBは増加した．HACではLgの場合，pHがplから離れる（pHが高くなる）に従ってIRとBは減少した．RNaseAの場合，IRはplから離れる（pHが低くなる）に従って増加したが，BはpHによらず一定で5前後であった．Lgに当量含まれるgeneticvariantsLgAとLgBは陰IECでは分離できたがHACおよび陽IECでは分離できなかった・HACは蛋白質に対してIECとは異なる分子認識をしていることが推察された・HACにおけるリガンドの状態についても考察した．

Six piston cores were collected along the NW–SE line in the Okinawa Trough close to the Chinese continental shelf edge and dated by 23 AMS 14 C measurements, along with planktonic δ 18 O stratigraphy and an ash layer dated as ∼6.3 ka. On a time scale to ∼19 ka, the ratio of total organic carbon to total nitrogen concentration (C/N ratio) displays gradual decreases toward the present and suggests the decrease of vascular plant fragment input to the trough from continental rivers. This suggestion is confirmed by similar decreases of lignin phenol content and ratio of C 28 fatty acid to C 16 fatty acid content in core RN93-PC3. The gradual decrease of land plant fragment input is interpreted to be a consequence of the retreat of the continental coastline during the deglacial period.

The mixing performance as well as the adsorption performance in expanded bed chromatography (EBC) was investigated by using various types of adsorption media (average particle size = 100–700 μm, density = 1100–1700 kg/m3, base matrix = hydroxyapatite, styrene-divinylbenzene, cross-linked agarose). The scale down study with 0.8 cm diameter columns was also attempted. Pulse response curves were measured with vitamin B12 as a tracer [Residence time distribution RTD experiments], and the HETP (height equivalent to a theoretical plate or plate height) values were calculated from the peak variance and the peak retention time. The HETP values for different types of packing media tested showed very similar values (0.5–1.0 cm), which did not depend on the flow-rate or the column diameter (0.8–2.6 cm). Dynamic binding capacity (DBC) values of lactic acid on a Dowex anion-exchange resin were determined from breakthrough curve (BTC) measurements for both EBC and fixed bed chromatography (FBC). The DBC values for EBC were similar to those for FBC. When the liquid feed contained insoluble particles (yeast cells) the degree of mixing increased. However, the contribution of the mixing to the total spreading of BTCs for EBC was usually small so that this increase in the mixing did not affect the adsorption performance or the DBC values significantly.

The purpose of this study is to present a case of chronic sclerosing dacryoadenitis with high level of IgG4 in a patient diagnosed earlier with Castleman disease. A 79-year-old man noticed a swelling of his lower left jaw that was first seen 8 years earlier. He was diagnosed with Castleman disease from the histopathological examination of a biopsy of the submandibular gland. Since then, the size of the gland had not changed, and he had no systemic inflammatory signs or symptoms. He developed diplopia a year earlier, and CT scans showed bilateral swelling of the lacrimal glands. He was referred to our hospital for further examinations. The patient underwent partial dacryoadenectomy. From the histopathological examinations, he was diagnosed with chronic sclerosing dacryoadenitis with high level of the serum IgG4. He underwent oral steroid therapy and the swollen lacrimal glands were significantly improved. The results suggest that there may be pathological links between IgG4-related dacryoadenitis and Castleman disease.

To compare the surgical outcomes during pars plana vitrectomy (PPV) for diffuse diabetic macular edema (DME) with pretreatment to those without pretreatment.The medical charts of 43 eyes of 46 patients who underwent PPV for DME were reviewed. The previously treated (Prev. Tx) group included 19 eyes of 20 patients who were pretreated with intravitreal bevacizumab, sub-Tenon injection of triamcinolone acetonide, and/or subthreshold micropulse diode laser photocoagulation before the PPV. The treatment naïve (Tx.Naïve) group included 24 eyes of 26 patients who underwent PPV alone. The central macular thickness (CMT) and best-corrected visual acuity (BCVA) were measured before treatment and 1, 3, and 6 months after.In both groups, BCVA at 3 and 6 months and CMT at 1 month or later were significantly better than preoperative visual acuity. CMT at 1 month or later significantly decreased from the preoperative value (p0.05). The differences in the BCVA and CMT between the Prev. Tx and Tx.Naïve group were not significant.PPV either with or without preoperative treatments can significantly improve the BCVA and reduce the CMT in patients with diffuse DME.

The tetra methyl ammonium hydroxide (TMAH) method has advantages of low consuming of time and sample in comparison with the other chemical analysis methods of organic compounds such as the alkaline CuO oxidation method. In this study, I aimed at a basic i

Purpose: It has been hypothesized that the increase in the b-wave during light adaptation is directly related to the level of cone malfunction in patients with retinitis pigmentosa (RP). Because this hypothesis has important bearing on the mechanism for the increase in the electroretinogram (ERG), we examined the increase in the amplitude of the cone ERG during light adaptation in patients with typical RP. Methods: Cone ERGs were recorded to Ganzfeld white flash stimuli in the presence of white background illumination in 51 RP patients and in 27 normal subjects. Results: In the normals, the increase in the b-wave amplitude during light adaptation ranged from 14–92% of the dark-adapted amplitude. All RP patients showed an amplitude increase that ranged from 5 to 100% of the baseline amplitude. This increase was not significantly different from that of the normals (p=0.71, unpaired t-test). The baseline amplitudes and the increase in the relative amplitude were weakly correlated in the RP patients (r=0.31; p=0.029). No significant difference was observed in the amplitude increase between patients with near normal b-wave implicit times and those with delayed times (p=0.17, unpaired t-test). Changes of the b-wave implicit time were not significantly different from those in the controls. Conclusion: These findings that the changes in the cone ERG with light adaptation in the RP patients were very similar to those in normal subjects do not support the proposed hypothesis that the increase in the b-wave amplitude during light adaptation was directly related to the level of cone malfunction.

Interleukin (IL)-4, IL-10, and IL-13, Th2 cell–derived cytokines, play major roles in the pathophysiology of allergic diseases. These cytokines up-regulate or down-regulate the production of arachidonic acid metabolites. In this study, we have investigated the effect of IL-4, IL-10, IL-13, and other cytokines on A23187-stimulated synthesis of leukotriene (LT) B4 in human polymorphonuclear leukocytes (PMNs). Production of LTB4 was measured by specific radioimmunoassay and high performance liquid chromatography. Messenger RNA (mRNA) expression of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and LTA4 hydrolase, which were involved in the synthesis of LTB4, was determined by reverse transcription–polymerase chain reaction and Northern blot analysis. Protein synthesis of their enzymes was determined by Western blot analysis. IL-4 and IL-13 enhanced A23187-stimulated LTB4 synthesis and increased mRNA expression and protein synthesis of LTA4hydrolase, but not those of cPLA2 or 5-LO. These results indicate that IL-4 and IL-13 transcriptionally or post-transcriptionally up-regulate the synthesis of LTB4, a potent chemotactic factor to PMNs, at the enzyme level of LTA4 hydrolase, and this up-regulation mechanism may participate in the development of allergic inflammation.

Both molecular recognition and transport phenomena play important roles in very high resolution ion-exchange (electrostatic interaction) chromatography (IEC) of proteins. A fast and simple method for obtaining important information on molecular recognition, peak retention (and the number of binding sites), and peak resolution of proteins from linear gradient elution experiments (salt concentration is increased linearly at a fixed mobile phase pH) is described. The proposed method was verified with β-lactoglobulin A and B (LgA, LgB) as model proteins. The gradient elution experimental data were obtained over a wide range of mobile phase pH with various types of IEC media. The number of binding sites involved in the retention (adsorption) decreased as the mobile phase pH approached the isoelectric points pI (= 5.1–5.2). However, even at pH 5.2 both LgA and LgB were retained on anion- and cation-exchange chromatography columns. The separation (resolution) of LgA and LgB became better when the pH approached t...

Silicone oil has been used to fill the vitreous cavity for long-term or permanent internal tamponade in eyes with proliferative vitreoretinopathy or complicated retinal detachment due to ocular trauma, giant retinal tears, proliferative diabetic retinopathy, and cytomegalovirus retinitis. Reports from the Silicone Study confirmed its efficacy in the treatment of proliferative vitreoretinopathy and addressed outcome differences in vitrectomized and nonvitrectomized eyes, combined retinotomy, silicone oil removal, and complications associated with silicone oil tamponade, such as intraocular pressure abnormalities and corneal abnormalities. Because silicone oil is lighter than water and not adequate in supporting the inferior quadrants, several heavier-than-water materials have been introduced for intraocular tamponade. Silicone oil can be a potential vehicle for delivering antiproliferative agents to treat proliferative vitreoretinopathy.

Eicosanoid receptors exhibit a highly conserved ERY(C)XXV(I)XXPL sequence in the second intracellular loop. The carboxyl end of this motif contains a bulky hydrophobic amino acid (L,I,V, or F). In human thromboxane A2 receptor (TXA(2)R), phenylalanine 138 is located at the carboxyl end of this highly conserved motif. This study examined the function of the F138 in G protein coupling. F138 was mutated to aspartic acid (D) and tyrosine (Y), respectively. Both mutants F138D and F138Y showed similar ligand binding activity to that of the wild type TXA(2)R. The Kd and Bmax values of either mutant were comparable to those of the wild type receptor. However, both mutants showed significant impairment of agonist induced Ca(2+) signaling and phospholipase C activation. These results suggest that the F138 plays a key role in G protein coupling.

Purpose: To evaluate the properties of the retina of a Japanese patient with enhanced S-cone syndrome by analyzing electroretinograms (ERGs) and visual evoked potentials (VEPs) elicited by different spectral stimuli. Methods: Ganzfeld spectral flashes in the presence of strong white adapting background illumination were used to elicit cone ERGs and VEPs. Results: The cone ERG elicited in the patient by short wavelength stimuli was distinctly different from the normal S-cone ERG. The action spectrum of the cone ERG confirmed its relative hypersensitivity to short wavelengths. The action spectrum of the VEP for the patient showed a similar relative hypersensitivity to short wavelengths. The response of the VEPs to short wavelength stimuli was different in waveform from the VEP response to longer wavelength stimuli observed in a normal subject. Conclusions: These results indicate that the hypersensitivity to short wavelengths is transmitted to the central nervous system and that there is a short wavelength transducing photopigment in many of the photoreceptors, either abnormal S-cones or photopic rods.

The retention and the resolution of β-lactoglobulin A and B (LgA, LgB) were investigated with various ion-exchange chromatography media. The number of sites involved in the retention (adsorption) decreased as the mobile phase pH approached the isoelectric points pI (=5.1–5.2). However, even at pH 5.2 both LgA and LgB were retained on anion- and cation-exchange chromatography columns. The separation (resolution) of LgA and LgB became better when the pH approached the pI in anion-exchange chromatography columns where the number of adsorption site values are small (ca. 2–3). The two proteins were not separated on cation-exchange chromatography columns. Factors affecting the resolution and the retention near the pI were discussed.

Very fine separation of proteins by stepwise elution ion-exchange chromatography is very often a unstable process. To characterize the unstability of such processes the elution volume variations were examined by the model equation which contained the ion-exchange capacity and the number of adsorption sites. The data needed for the model calculation were obtained from gradient elution experiments. As a model separation system stepwise elution of a model protein (beta-lactoglobulin) near the isoelectric point on a weak cation-exchange chromatography column was chosen. The elution volume varied significantly with a small change in the ion-exchange capacity. It was found that the ionic strength of the elution buffer must be adjusted in order to compensate a change in the elution volume due to the ion-exchange capacity variations. The ionic strength and the pH of the elution buffer were also found to be important variables affecting the elution volume. In this model separation system, it was indicated that the pH should be within +/-0.1 unit and the ionic strength within +/-0.002 mol/l in order to meet the criteria (+/-5% elution volume variation). It is recommended that gradient elution data be obtained for predicting elution volume variations in stepwise elution. By using the gradient elution data the process diagnosis can be performed, and the important information on the process stability can be obtained.

Cone electroretinograms (ERGs) elicited by different chromatic stimuli were recorded from patients with typical retinitis pigmentosa (RP) younger than 50 years of age. Ganzfeld color flashes on a bright white background illumination were used to elicit short-wavelength-sensitive (S-), and mixed long- (L-) and middle- (M-) wavelength-sensitive cone ERGs. Three patients with dominant inheritance, 22 patients with non-dominant inheritance, and 27 age-similar normal subjects were compared. Although the b-wave amplitudes of both the S- and L,M-cone ERGs were reduced in the RP patients, the S-cones were reduced to a greater degree than the L,M-cones. No significant difference in the S-cone reduction was observed between patients with dominant inheritance and those with non-dominant inheritance. The selective reduction of the S-cone system, reported previously in psychophysical studies, may be attributable not only to the macular region but also to the entire retina.

Human leukemia (HL) 60 cells were differentiated by dimethylsulfoxide (DMSO) treatment to granulocyte-like cells, leukotriene (LT) synthesizing activity of which was increased in response to the differentiation of the cells. Four synthesizing enzymes, cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), LTA4 hydrolase and LTC4 synthase, and an enzyme associated protein, 5-lipoxygenase activating protein (FLAP) are involved in the generation of LTC4 and LTB4. We examined the expression of messenger RNA (mRNA) for these LT synthesizing enzymes and an associated protein in DMSO differentiated HL-60 cells by reverse transcriptase polymerase chain reaction (RT-PCR). The production of LTC4 and LTB4, measured by radioimmunoassay (RIA), was increased after the incubation with DMSO for more than 3 days. Messenger RNA abundance for 5-LO, LTC4 synthase and LTA4 hydrolase was increased, that for FLAP was stable, but that for cPLA2 was decreased. These results indicate that DMSO induced increase of LT synthesis is associated with the increase of mRNA expression of 5-LO, LTC4 synthase and LTA4 hydrolase, although the precise regulatory mechanisms of the increased mRNA expression are not determined. We also investigated an action of dexamethasone (DEX) on DMSO-induced enhancement of LT synthesis. DEX suppressed DMSO induced increase of LTC4 synthesis, but rather enhanced DMSO induced LTB4 production. The DEX attenuated the DMSO-induced increase of mRNA expression for LTC4 synthase, but showed no effect on that for LTA4 hydrolase. The inhibition of LTC4 synthesis is associated with the suppression of mRNA expression for LTC4 synthase. However, increased LTB4 synthesis by DEX is regulated by the mechanisms which are independent from mRNA level of LTA4 hydrolase.

Cone electroretinograms (ERGs) in response to different color flashes were examined using a Ganzfeld stimulus in 19 eyes after successful retinal detachment surgery. In the operated eyes, the short wavelength sensitive (S-) cone b-wave was reduced more than the mixed long (L-) and middle (M-) wavelength sensitive cone b-waves. The ratio of the S-cone ERG b-wave amplitude between operated eyes and fellow eyes was significantly lower than the L- and M-cone ERG b-waves (P < .01). These ERG results indicated that the S-cone system is more impaired than the L- and M-cone systems after retinal detachment surgery.

There are substantial numbers of reports showing that leukotrienes (LTs) play important roles in adult asthma. No definite evidence has been demonstrated that LTs are involved in asthma attacks in children, although it is highly expected. In this report, we demonstrated that the levels of LTB4 and LTC4 but not thromboxane B2 (TXB2), a stable metabolite of TXA2, were significantly elevated in the bronchoalveolar lavage fluid, which was obtained from intubated and mechanically ventilated children with severe asthma attacks. This is direct evidence that LTB4 and LTC4 predominantly participate in asthma attacks in pediatric patients.

The short wavelength-sensitive (S-) cone electroretinogram (ERG) is selectively reduced in diabetic patients both with and without retinopathy, but the exact mechanism of the vulnerability of the S-cone system is still unclear. This study examined relationships of the S-cone ERG to systemic factors in diabetes. Cone ERGs to different color flash stimuli were examined in the presence of bright white background illumination in 17 diabetic patients without retinopathy and in 17 diabetics with background retinopathy. Relationships of the amplitude and implicit time of the S-cone ERG to the following systemic factors were statistically analyzed: patients' age, hemoglobin A1 level, method of diabetic control, presence of retinopathy, and presence of nephropathy. The amplitude of the S-cone ERG b-wave was significantly reduced in diabetics treated with insulin and in those associated with nephropathy. No significant correlation was found between the S-cone ERG and patient's age, hemoglobin A1 level and presence or absence of retinopathy. A selective reduction of the S-cone ERG is observed in patients whose metabolic control has been poor for a longer period, suggesting that microvascular changes may play a role in the S-cone ERG impairment.

Background Cytokines, such as interleukin (IL)-5, produced by T helper type 2 (Th2) cells appear to play an important role in the inflammatory processes associated with atopic dermatitis. The roles of cytokines produced by Th1 cells remain controversial. Objective We examined IL-5 and IL-2 mRNA abundance in and protein production by peripheral blood mononuclear cells (PBMCs) from patients with atopic dermatitis and compared with those from controls. Methods Peripheral blood mononuclear cells were isolated from six children with atopic dermatitis and six control children, and stimulated with both phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA). The abundance of IL-5 and IL-2 mRNA in PBMCs was measured by reverse transcription-polymerase chain reaction analysis. The production of IL-5 and IL-2 by PBMCs was also determined by enzyme-linked immunosorbent assay. Results After incubation with PHA and PMA, PBMCs from atopic children showed significantly higher IL-5 mRNA abundance (P .05) and IL-5 production ( P .01) as well as a lower amount of IL-2 mRNA ( P = .056) and IL-2 production (P .05) than those from healthy controls. The time course of changes in IL-5 mRNA abundance induced by PHA and PMA in PBMCs from atopic children differed markedly from that observed with healthy controls, whereas the time course of changes in IL-2 mRNA abundance were similar between the two groups. Conclusions The increased IL-5 and decreased IL-2 production observed with PBMCs from children with atopic dermatitis may underlie the activation of eosinophils and high serum immunoglobulin E concentrations also apparent in such individuals. An imbalance in the number and activity of Th1 and Th2 cells is likely to be responsible for the abnormal pattern of cytokine production in atopic dermatitis.

• Background: Aminoaciduria is found in Lowe's syndrome. No studies of concentrations of ascorbic acid and amino acids in the aqueous humor of the syndrome have been performed. We examined these concentrations in a patient with Lowe's syndrome. • Methods: Ascorbic acid and amino acid levels in the aqueous humor and plasma of a male infant were measured by means of high-performance liquid chromatography. The patient, who had congenital cataract, miotic pupils, opaque corneas, glaucoma, aminoaciduria, normal levels of ascorbic acid and amino acid in the plasma, and renal tubular acidosis, underwent trabeculotomy, lensectomy, and anterior vitrectomy in both eyes. • Results: Intraocular pressure in both eyes decreased to within the normal range, but both corneas remained opaque. The amino acid levels in the aqueous humor were similar to those in the plasma, but intracameral ascorbic acid levels were decreased. After topical instillation of ascorbic acid, the corneas became transparent. The proband's mother had good visual acuity but paracentral lens opacities in both eyes. His maternal grandmother had scattered cortical opacities in both lenses. • Conclusion: In this infant with Lowe's syndrome, we found intracameral levels of amino acids similar to those in the plasma. Levels of ascorbic acid in the aqueous humor were decreased.

We examined the action of Shinpi-To (Formula divinita; TJ-85), a granular extract of seven Chinese medicinal herbs that is used in treating childhood asthma, on the leukotriene synthesis in rat basophilic leukemia-2H3 cells (RBL-2H3 cells). IgE-loaded cells were stimulated with anti-IgE serum in the presence or absence of Shinpi-To. Released LTC4 and LTB4 were measured by radioimmunoassay (RIA). Shinpi-To significantly inhibited IgE-mediated synthesis of leukotriene (LT)C4 and LTB4. To identify the inhibitory sites, we investigated the action of this extract on four synthetic enzymes, phospholipase A2 (PLA2), 5-lipoxygenase (5-LO), LTC4 synthase, and LTA4 hydrolase. Shinpi-To inhibited the A23187-stimulated release of [3H]arachidonic acid (AA) from the cell membrane, reflecting an effect on PLA2 activity. It also suppressed production of LTC4 and LTB4 when cell lysates were incubated with AA as substrate. It did not inhibit the production of LTC4 and LTB4 when LTA4-free acid was used as the substrate. Shinpi-To did not inhibit the IgE-mediated increase of intracellular Ca2+ ([Ca2+]i) concentration. Results indicate that Shinpi-To inhibits LT synthesis by inhibiting PLA2 and 5-LO activities without affecting the mobilization of [Ca2+]i.

A series of reports for optimal procedures for inhalation technique with beta-2 metered dose inhaler (β2-MDI) were previously published in this journal by a research group for β2-inhalation therapy in children in western Japan. We have surveyed the related issues for inhalation techniques of β2-MDI from special ists for child asthma in all over Japan and general pediatricians in Fukuoa-city and Saga-prefecture. The results demonstrated that (1) 93% of allergy specialists and 82% of general pediatricians prescribed β2 MDI with instructions for use, (2) only 28% of allergy specialists, however, confirmed the procedures by letting patients inhale MDI in front of doctors and (3) not many doctors in both groups instructed the in halation techniques which were recommended as the most optimal procedures by the research group.

The mechanism of the separation of galactosylkojic acid (GKA) produced by the reaction of immobilized β-galactosidase with kojic acid (KA) and lactose as substrates using Toyopearl HW40 size exclusion chromatography was investigated. The effects of the mobile phase conditions (salt concentration and pH) on the distribution coefficient K for GKA and KA determined from the peak retention volume were investigated. The K values for GKA and KA decreased slightly when the pH in the mobile phase was increased from 6 to 7, while a further increase in the pH from 7 to 8 caused a considerable decrease in the K values. The K values increased with increasing NaCl concentration (0 to 0.5 M), especially at pH 8. The K value for GKA at pH 8 was almost the same as that for neutral sugar having the same molecular weight. As the gels have negative charges at pH 6–8 and the dissociation constant of KA is ca. 10 −8 , these results indicate that the distribution of GKA and KA is governed by hydrophobic interaction adsorption and electrostatic repulsion. Although complete baseline separation of GKA was achieved under any of the conditions examined, on the basis of the plate height and K value data the optimum mobile phase conditions were determined to be pH 7 and 50 mM NaCl.

Practical considerations lead to the use of small columns in preparative chromatography. In these applications extra column effects may overwhelm all broadening effects. The influence of the extra column effects on the band broadening and migration is demonstrated by simulation. The model assumes linearity of extra column effects. Consequently, these effects could be described by the convolution of the initial applied band width, an exponential decay and Gaussian broadening. An attempt has been made to discriminate between the column, pre- and postcolumn broadening.

Separation of acetic acid from palm oil mill effluent (POME) to increase its concentration by an anion exchange resin was examined as a preliminary study for its recovery from POME that had been anaerobically treated by sludge from a palm oil mill. This paper concerns the acetic acid thus separated for producing bacterial polyhydroxyalkanoate (PHA) by Alcaligenes eutrophus. It was found that sludge particles in POME strongly inhibited the adsorption of acetic acid on the anion exchange resin. Removing the sludge particles from the POME facilitated the separation of acetic acid from the POME efficiently. The concentrated acetic acid thus obtained from anaerobically treated POME could be used as a substrate in the fed-batch production of polyhydroxyalkanoate by Alcaligenes eutrophus.

To evaluate the heat effects of radiofrequency ablation (RFA) on normal bone by mechanical testing, MRI, and histology. The institutional animal care and use committee approved the animal study. Thirty-two adult Japanese white rabbits were included in our study. Bone biopsy needles were inserted from the distal end of the right (RFA side) and the left (control side) femurs using a fluoroscopic guide. A 17-gauge internally cooled RFA electrode with a 2-cm active tip was inserted through the needle to the right femur, and RFA was performed for 12 min using a 200-W generator. Animals were divided into four groups and 8 animals from each group were euthanized on day 1, day 7, day 30, and day 60 after RFA. MRI was performed prior to euthanasia. Three-point bending test was performed to measure flexural strength. Student’s t test was used to evaluate for significant differences between RFA and control side for each group. Femurs underwent histological examination by hematoxylin and eosin staining after the bending test. MRI showed a high-intensity rim around the bone on T2WI. Three-point bending test showed no statistically significant differences (P

To examine whether c-Fos, phosphorylated c-Jun (p-c-Jun), members of transcriptional factor activator protein 1 (AP-1) family and phosphorylated c-Jun N-terminal kinase (p-JNK) are associated with neuronal degeneration in retinas of diabetic patients.Retinal cryosections from five pairs of normal and five pairs of diabetic human eyes were immunostained for c-Fos, p-c-Jun and p-JNK followed by costaining with Fluoro-Jade B (FJB), a marker for identifying degenerative neurons. Additionally, cells were stained with 4, 6-diamidino-2-phenyl indole (DAPI) to facilitate counting the total number of cells. The number of c-Fos, p-c-Jun and p-JNK positive cells costained with FJB was assessed in the ganglion cell layer (GCL) together with the total number of DAPI-positive cells.The number of FJB-positive cells in the GCL of diabetic retinas was significantly increased compared to those of non-diabetic retinas. The GCL of diabetic retinas, compared to those of the non-diabetic retinas, showed increased number of c-Fos, p-c-Jun and p-JNK-positive cells that coexisted with FJB-positive signals.This study indicates that increased expression of c-Fos, p-c-Jun, members of AP-1 transcriptional factor and p-JNK is associated with neuronal degeneration in the GCL of retinas in diabetic patients.

Background To present our findings in a case of Werner syndrome with refractory cystoid macular edema (CME) and to determine the expression and the distribution of WRN proteins in human retinas. Case presentation A 35-year-old man with Werner syndrome who developed CME after YAG laser treatment was studied. Optical coherence tomographic (OCT) scans were used to examine the CME in the right eye. The patient received topical eye drops (0.1% bromfenac sodium hydrate twice daily and 1% dorzolamide hydrochloride thrice daily), sub-Tenon triamcinolone injection thrice, intravitreal bevacizumab injection twice, and pars plana vitrectomy of the right eye. Genetic analyses were performed to diagnose the disease. To examine the expression and distribution of WRN proteins in the retinas, immunohistochemistry for WRN proteins was performed in human retinas. The CME in the right eye was not improved by any of the treatments. During the follow-up period, CME developed in the left eye. Genetic analyses detected compound heterozygosity, Mut4 and Mut11, in the WRN gene and the individual was diagnosed with Werner syndrome. Immunohistochemical analysis of WRN proteins expression in human retinas showed that WRN proteins were expressed in the parts of the Müller cells in the inner nuclear layer and outer nuclear layer. Conclusion Patients with Werner syndrome can develop severe CME after laser treatment. A pathological link may exist between mutations in the WRN gene and the development of CME in patients with Werner syndrome.

Purpose To investigate the association of the Fourier domain optical coherence tomography (FD-OCT, Optovue) parameters with the level of HbA1c and duration of the diabetes mellitus (DM) in patients at the early stage of diabetic retinopathy (DR). Methods Thirty eyes of 30 patients with minimal DR (non-DR or mild NPDR) that were examined at the Chiba University Hospital from December 2011 to March 2012 were studied. The participants had no other ocular diseases and no history of surgeries. Three parameters embedded in the Optovue, the macular mapping 5 (MM5), RNFL, and ganglion cell complex (GCC), were performed, and the coefficients of correlation between these parameters and the HbA1c and the DM duration were analyzed with the Spearman rank correlation. A P

The purpose of this study was to determine the effect of low concentrations of advanced glycation end-products on neurite regeneration in isolated rat retinas, and to determine the effects of neurotrophin-4 on regeneration in advanced glycation end-products exposed retinas. Retinal explants of 4 adult Sprague-Dawley rats were cultured on collagen gel and were incubated in; (1) serum-free control culture media, (2) glucose-advanced glycation end-products–bovine serum albumin media, (3) glycolaldehyde-advanced glycation end-products–bovine serum albumin media, (4) glyceraldehyde-advanced glycation end-products–bovine serum albumin media, (5) glucose-advanced glycation end-products+neurotrophin-4 media, (6) glycolaldehyde-advanced glycation end-products+neurotrophin-4 media, or (7) glyceraldehyde-advanced glycation end-products+neurotrophin-4 supplemented culture media. After 7 days, the number of regenerating neurites from the explants was counted. Then, explants were fixed, cryosectioned, and stained for TUNEL. The ratio of TUNEL-positive cells to all cells in the ganglion cell layer was determined. Immunohistochemical examinations for the active-form of caspase-9 and apoptosis-inducing factor were performed. In retinas incubated with advanced glycation end-products containing media, the number of regenerating neurites were fewer than in retinas without advanced glycation end-products, and the number of TUNEL-positive cells and caspase-9- and apoptosis-inducing factor-immunopositive cells was significantly higher than in control media. Neurotrophin-4 supplementation increased the numbers of regenerating neuritis, and the number of TUNEL-positives, caspase-9-, and apoptosis-inducing factor-immunopositive cells were significantly fewer than that in advanced glycation end-products without neurotrophin-4 media. Low doses of advanced glycation end-products impede neurite regeneration in the rat retinas. Neurotrophin-4 significantly enhances neurite regeneration in retinas exposed to advanced glycation end-products.

Purpose: Hyperglycaemia has been identified as major risk factor for diabetic retinopathy (DR). It is widely accepted that the progression of DR is mainly due to a local imbalance of pro- versus anti-angiogenic factors in the retina. In this study, we investigated whether retinal pigment epithelial (RPE) cells produced pro-angiogenic factors under high glucose (HG) conditions in vitro. Methods: Cultured human retinal endothelial (RE) cells were exposed to conditioned medium from retinal pigment epithelium cells (ARPE-19) grown in HG medium and assessed for tube formation. Based on the expression profiles of ARPE-19, we investigated whether ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions using cultured human RE cells as the test system for experiments with recombinant protein, conditioned medium from ARPE-19 and RNA interference (RNAi). Results: The conditioned medium from ARPE-19 cultured under HG conditions promoted tube formation of cultured human RE cells. GeneChip analysis showed that ANGPTL4 was one of the highest upregulated genes under HG conditions. In addition, recombinant ANGPTL4 promoted all of the elements of angiogenesis in human RE cells in vitro. The results of experiments using conditioned medium from ARPE-19 combined with RNAi demonstrated that ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions. Conclusions: ANGPTL4 was induced by high glucose in RPE cells and exhibited potent angiogenic activity on RE cells. Our results are unique and may potentially add a new candidate to the long list of molecules involved in diabetic retinopathy.