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To determine the characteristics of the laterality of the ocular microcirculation parameters obtained from laser speckle flowgraphy (LSFG) in the optic nerve head (ONH).We reanalyzed a total of 240 healthy subjects (169 men, 71 women) who had participated in a medical checkup program. We analyzed the average mean blur rate (MBR), maximum MBR (Max-MBR), minimum MBR (Min-MBR), and area ratio of the blood stream (ARBS). As the pulse waveform parameters, we also calculated the skew, blowout score (BOS), blowout time (BOT), rising rate, falling rate, flow acceleration index (FAI), acceleration time index (ATI), resistivity index (RI), and beat strength over MBR (BOM). All parameters were compared between left and right eyes.MBR-average, MBR-Max, MBR-Min, ARBS, skew, BOT, rising rate, falling rate, FAI, ATI, and ARBS did not differ significantly between the right and left eyes. The BOS in the right eyes was significantly lower than that of the left eyes, and the RI and BS in the right eyes were significantly higher than those of the left eyes. Similarly, for the whole of the ONH, the BOS in the tissue area (Tissue) and in the vessel area (Vessel) of the right eyes were significantly lower than those of the left eyes, and RI-Tissue, RI-Vessel, BOM-Tissue, and BOM-Vessel in the right eyes were significantly higher than those of the left eyes.By using LSFG, we observed laterality of the BOS, RI, and BOM, all of which are pulse wave form parameters in the ONH.

Background The effect of various reduced doses of verteporfin photodynamic therapy (PDT) on choroidal blood flow in chronic central serous chorioretinopathy (CSC) remains unclear. Therefore, this study aimed to evaluate choroidal blood flow after half-dose PDT (1/2PDT) and one-third dose PDT (1/3PDT) with verteporfin for chronic CSC using laser speckle flowgraphy and spectral-domain optical coherence tomography. Methods Twenty-seven eyes of 27 patients with serous retinal detachment (SRD) caused by chronic CSC for more than 6 months were included in this study. Patients were divided into the 1/2PDT (n = 12; January 2018 to July 2019) and 1/3PDT (n = 15; August 2016 to December 2017) groups based on the treatment period. The best-corrected visual acuity (BCVA), central retinal thickness (CRT), central choroidal thickness (CCT), and mean blur rate in the macular area (m-MBR) and optic nerve head (ONH-MBR) were obtained using laser speckle flowgraphy and evaluated at baseline (pre-treatment), and 2 weeks, 1 month, 3 months, and 6 months after treatment. Results We found that SRD disappeared after 1 month in 92 and 93% of patients’ eyes in the 1/2PDT and 1/3PDT groups, respectively. Recurrence of SRD was observed in one eye at the 6-month follow-up after 1/2PDT and two eyes at the 3-month follow-up after 1/3PDT. No significant improvement was observed in baseline BCVA in the 1/3PDT and 1/2PDT groups. The average m-MBR against baseline significantly decreased at 2 weeks and 1, 3, and 6 months in the 1/2PDT group. The average m-MBR against baseline decreased significantly only at the 2 weeks follow-up in the 1/3PDT group. The average rate of change in the CCT against baseline decreased significantly throughout for up to 6 months in the 1/2PDT group and for up to 3 months in the 1/3PDT group. No significant fluctuation was observed in the ONH-MBR. Conclusions: Here, PDT significantly affected choroidal blood flow depending on the verteporfin dose in chronic CSC. Trial registration This trial was retrospectively registered (UMIN000026850; Approval date 03/04/2017).

Potential differences in the nature of the influences of aging and gender on the optic nerve head (ONH) microcirculation, using laser speckle flowgraphy (LSFG) were evaluated. We studied 908 healthy subjects (men = 701, age: 50.0 ± 9.1 yrs, women = 208, 49.8 ± 9.5 yrs, p = 0.76). The average, maximum (Max), and minimum (Min) mean blur rate (MBR) in a heartbeat were evaluated. The parameters were analyzed separately for the tissue, vessels, and throughout the ONH (All). We investigated which MBR sections are correlated with gender and age by univariate and multivariate regression analyses. The Max MBR-All (r = −0.31) was most strongly correlated with gender (men = 1, women = 0). The Min MBR-All (r = −0.24) was most strongly correlated with age, followed by Min MBR-All (r = −0.20). The factors contributing independently to the Max MBR-All were gender (β = −0.15), pulse pressure, spherical refraction, ocular perfusion pressure, and red blood cell (RBC) count. The factors contributing independently to the Min MBR-Vessel were gender (β = −0.09), age (β = −0.25), body mass index, heart rate, and spherical refraction. The factors contributing independently to the Min-MBR-All were age (β = −0.22), heart rate, and RBC count. Our results revealed that gender differences influence the Max MBR, and aging influences the Min MBR. These correlations were stronger than that of average MBR.

Purpose: To determine a correlation between temperature and blood flow in the ocular anterior segment, and their effects on corneal temperature. Methods: In experiment 1, we recruited 40 eyes and measured the temperature and blood flow in the ocular anterior-segment (upper/lower eyelid skin, palpebral and bulbar conjunctiva, and cornea) before and after application of warm compresses. In experiment 2, we recruited 20 eyes and measured the same tissues before and during stimulation using water and capsaicin solution in the oral cavity. Results: In experiment 1, the temperatures of the upper/lower eyelid skin and cornea increased significantly until 15 min after the application of the warm compress, the temperatures of the palpebral and bulbar conjunctiva increased significantly until 10 min. The blood flow in the upper/lower eyelid skin and bulbar conjunctiva increased significantly until 10 min, and that of the palpebral conjunctiva increased significantly until 15 min. In experiment 2, the temperatures were correlated significantly with the blood flow in the upper and lower eyelid skin and palpebral and bulbar conjunctiva. The temperature of all locations and palpebral conjunctival blood flow contributed independently to the corneal temperature. Conclusions: In the ocular anterior segment, the temperature and blood flow were correlated significantly, and contributed to the corneal temperature.

Metagenomic analysis is the comprehensive study of DNA using clinical specimens of organisms including bacteria, fungi, and viruses. In this study, we investigated the efficacy of metagenomic analysis for diagnosing ocular infections, including 11 keratitis cases, four iridocyclitis cases, and one endophthalmitis case. Corneal scraping, aqueous humor, and vitreous humor, were collected respectively. Ocular specimens were used for bacterial and fungal culture, and PCR for detecting viral DNA. Shotgun metagenomic sequencing for 150 bases of single end was performed by Illumina MiSeq® System. Sequence was retrieved from the database at NCBI using a MegaBLAST search. Since Propionibacterium spp. are commensal bacteria found at the ocular surface, they were excluded from analysis. Six cases (37.5%) were positive for culture or PCR. Metagenome techniques revealed that 9 cases (56.3%) included genomes of organisms that were considered pathogenic in specimens. Five cases (31.3%) possessed genomes of organisms like themselves that were detected by culture and PCR. Six cases (37.5%) were negative for culture, PCR, and metagenome analysis. Moreover, viral pathogens (HSV-1, 2 cases, and VZV, 1 case) were detected by only metagenome analysis. Metagenome analysis using an ocular sample can detect microbial genome comprehensively, and viral pathogens, which were not detected by conventional examination.

Production of NO may be regulated by argininosuccinate synthetase and argininosuccinate lyase which recycle citrulline to arginine, and by arginase which hydrolyzes arginine to urea and ornithine. Expression of these and related enzymes in rat eye was studied.mRNAs for the enzymes were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Localization of the enzyme proteins and mRNAs was analyzed by immunohistochemistry and in situ hybridization, respectively.In RT-PCR analysis, arginase II (nonhepatic type) mRNA was detected in retina and weakly in cornea, whereas arginase I (hepatic type) mRNA was not detected in any area. mRNAs for argininosuccinate synthetase, argininosuccinate lyase, ornithine aminotransferase and ornithine decarboxylase were present in all areas. In immunohistochemical analysis, arginase II was stained in cornea, epithelium of iris and ciliary process, inner part of neural retina and retinal pigment epithelium. Immunostaining of ornithine aminotransferase resembled that of arginase II. In in situ hybridization, arginase II and ornithine aminotransferase mRNAs were located much as seen with the enzyme proteins.These results indicate that arginine recycling activity from citrulline is present widely in ocular tissues, whereas expression of arginase II differs among the tissues. We suggest that NO production may be regulated by these enzymes in the cells where NO synthase is colocalized. Colocalization of arginase II and ornithine aminotransferase suggests a role for arginase II in collagen synthesis in the eye.

医療用材料 (特に創傷被覆材) として有用なポリウレタンフィルムの作製と物性測定を行った. ポリウレタンの合成は, ジイソシアナートとして毒性がより低いと考えられるbis(4-isocyanatocyclohexy1) methaneを用い, 触媒の存在下ポリオールを反応させ, イソシアナート基を末端に持つプレポリマーとした後, ジアミンと反応させ, ソフトセグメントとハードセグメントを併せ持つセグメント化ポリウレタンを得た. 工業的スケールアップ時でも, 実験室レベルで作製したポリウレタンフィルムと同様の物性を持つフィルムが得られた. ポリウレタンのポリマー構造式中に, ポリオール部をできるだけ多く含有させることにより, 極めて高い水蒸気透過性を持つポリウレタンフィルムが得られた.このフィルムは, 高水蒸気透過性以外に, 吸水時の寸法安定性が高いこと, 耐光性のあること, 機械的強度が高いこと, 伸展性のあること, 安全性の高いことなどの特徴があった.Lamkeらの「熱傷受傷時の皮膚創部表面から蒸散される水蒸気量」と,「適切なポリウレタンフィルムの厚さ」の二つの因子から, 創傷被覆材として使用し得るフィルムの選択を行った.

The mathematics developed properly in Japan during the Edo period (1603–1867) is called Wasan (Japanese Mathematics). Its roots are in the ancient Chinese mathematics which flourished in the Song and Yuan dynasties (962–1368), in particular, but the later developments in Japan have been thought to be independent of China or Europe. The author claims, however, that Wasan was influenced by Europe first through Chinese translations of European books starting with the Fundamentals for Astronomy (1629), which was purchased by a son of Tokugawa Ieyasu as early as 1632, and from the end of the eighteenth century on directly through books in Dutch.

Compared with the activity obtained with a high-protein diet in rats, a low-protein diet doubled the activity of ornithine aminotransferase [EC 2.6.1.13] (OAT), a key enzyme for citrulline synthesis, in the small intestine. The induction of ornithine aminotransferase in the small intestine by the low-protein diet and its suppression by the high-protein diet, and the converse in the liver, were immunohistochemically verified with anti-OAT antiserum. The immunohistochemical studies revealed that ornithine aminotransferase molecules localized in the villous surface epithelia, but not in the cryptic epithelia, were most responsive to the changes in dietary conditions, these results indicating that intestinal ornithine aminotransferase may be involved in the ornithine supply to the liver, with the reversal of the enzyme reaction occurring with a low-protein diet. Reconstituted model experiments on citrulline synthesis revealed that the addition of ornithine carbamoyl-transferase and carbamoyl phosphate was essential to overcome the unfavorable equilibrium of the reverse reaction, and the further addition of glutamate dehydrogenase and ammonia resulted in a stimulating effect.

The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process.

Native human cytosolic alanine aminotransferase (EC 2.6.1.2) was found to be a homodimer consisting of two 55 kDa subunits. The human enzyme was more cationic and more susceptible to heat inactivation and heavy metal-inhibition than its rat homologue. Isoelectric focussing separated three human isoforms and four rat isoforms of cALT that differed in their isoelectric points. Two subtypes of the enzyme which differed in apparent molecular weight on sodium dodecylsulfate polyacrylamide gel electrophoresis were detected in rat, the liver type and the muscular type. This microheterogeneity was not found for human cytosolic alanine aminotransferase. Immunohistochemical studies revealed that the rat liver enzyme was exclusively localized in periportal hepatocytes, consistent with its role in gluconeogenesis. In human hepatocytes the plasma membrane was intensely stained, implicating an intracellular localization near the plasma membrane.

There is increasing evidence for the role of the Fas/Fas ligand interaction in the immunoregulation of T-cells. We studied the expression of the Fas ligand (FasL) in activated peripheral T-cells in vitro, and its relation to autonomous cell death by flow cytometry. Following the stimulation of lymph node T-cells with anti-CD3 and rIL2, the mRNA level of FasL increased more than four times during the first 2 days over the level before stimulation. The surface expression of FasL was observed on 27% of the population at day 2 after stimulation and increased to approximately 50% at day 3. Kinetic analysis by flow cytometry, however, indicated that all T-blasts transformed during activation did not express FasL. FasL expression became evident simultaneously with the termination of cell expansion. Since cells remained viable (> 90%) at day 3 as judged by trypan blue-exclusion, cell membranes expressing FasL were supposed to be still intact. Concomitantly with FasL-expression, spontaneous DNA fragmentation was observed. These observations support the idea that autonomous Fas/FasL interaction mediates apoptosis in activated peripheral T-cells as demonstrated in T-cell hybridoma or established T-cells.

The Caenorhabditis elegans unc-18-encoded protein (UNC-18) is implicated in the processes of vesicle targeting, docking, and/or fusion. To further characterize the properties of this important neural protein, we expressed it at a high level in Spodoptera frugiperda Sf21 cells using a baculovirus expressing system. A cDNA containing the coding sequence for UNC-18 was inserted into the transfer vector pBlueBac to yield the recombinant virus pAcNPV/unc-18. At maximal expression, the recombinant virus produces a protein of 67 kDa, which constitutes about one-third of total cell protein. The UNC-18 protein was highly purified and its biochemical and functional properties were assessed. The protein is globular with an isoelectric point of 6.95. Circular dichroism spectroscopy indicated that the alpha-helix and beta-sheet account for 10.0 and 59.0%, respectively. Immunolabeling the Sf21 cells expressing UNC-18 showed that the expressed UNC-18 is predominantly localized in the cytoplasm as a soluble monomer. The protein is phosphorylated by protein kinase C and binds to the recombinant C. elegans syntaxin in vitro. These findings suggest that in vesicle traffic UNC-18 is a regulator factor associated with the plasma membrane through syntaxin, although intrinsically cytoplasmic.

The cDNA encoding ornithine aminotransferase (EC 2.6.1.13; OAT) was isolated from a human kidney cDNA library. The isolated cDNA contained the entire protein coding region and partial 3′- and 5′-untranslated regions. The nucleotide sequences of human kidney OAT cDNA were absolutely homologous with those of human liver OAT cDNA, and human kidney and liver OAT fused completely against the antibody to human kidney OAT in an Ouchterlony double diffusion test. These findings settled the controversy as to which characteristics of liver and kidney OAT isozymes are different. An N-terminal sequence analysis of purified mature human kidney OAT clarified that the leader peptide was cleaved between Gln-35 and Gly-36.

An inhibitor of mouse factor B was purified from lysate of L cells by a human factor B-affinity column. The purified inhibitor was found to be homogeneous with a molecular weight of about 25,000 and identical to the 25K protein isolated from L cell-lysate by the coprecipitation method with anti mouse factor B. Hemolytic titration of the activity and PAGE-analysis of the inhibitor-factor B complex showed that 1 mol of inhibitor reacted with 1 mol of mouse factor B and inactivated its hemolytic activity.

The cyclic guanosine 3′, 5′-monophosphate levels of cultured cells including CCL-6 cells and L cells were independent of heat-stable enterotoxin activity of Escherichia coli but elevated by heat-stable enterotoxin of Yersinia enterocolitica.

The relationship between invasiveness and calcium dependency was examined in various strains of Yersinia enterocolitica and Y. pseudotuberculosis by using established cell lines. Infection with calcium-dependent bacteria resulted in the formation of microvilli and the adherence of bacteria on the cell surface, and the adherent bacteria were ingested 1.5 hr after infection. Morphological changes in the cells became visible 2 to 3 hr after infection, and intracellular multiplication of the ingested bacteria was noted. When the cells were incubated with bacteria at 37 C for 1.5 hr and then at 25 C, however, the morphological changes in the infected cells were not observed. No isogenic strains that had lost calcium dependency for growth at 37 C were able to elicit the morphological changes in the cells, though they possessed the ability to adhere to and penetrate the cells. The antigen(s) supposedly related to cytotoxicity of the calcium-dependent Yersinia was sought by using antibodies prepared against calcium-dependent bacteria and then absorbed with calcium-independent bacteria and with calcium-independent bacterial cytosol. Double diffusion tests between the antisera and bacterial cytosol extracts revealed the presence of an antigen which was a cytoplasmic substance common to all calcium-dependent but not calcium-independent strains of Y. enterocolitica and Y. pseudotuberculosis.

proteins which were able to bind noncovalently with mouse factor B were found in cells that are nonsecretors of factor B such as mouse-established monocytic cells and L cells but not in peritoneal resident macrophages. These proteins were isolated from lysates of L cells and separated into four distinct proteins by preparative SDS-polyacrylamide gel electrophoresis, with molecular weights of 25K , 28K , 33K, and 35K . The individual proteins formed a complex with purified mouse factor B at a molecular ratio of 1: 1 and inhibited its hemolytic activity. Proteins 25K and 28K inhibited the hemolytic activity of an activated form of factor B combined with cobra venom factor as well as that of the native form. These inhibitors did not affect the hemolytic activity of the second component of complement in mouse serum. The inhibitory activity of the 25K protein was partially inhibited by antiserum raised against it in rabbits.