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1.  Anterior capsule contraction and intraocular lens decentration and tilt after hydrogel lens implantation 
The British Journal of Ophthalmology  2001;85(11):1294-1297.
AIM—To prospectively investigate changes in the area of the anterior capsule opening, and intraocular lens (IOL) decentration and tilt after implantation of a hydrogel IOL.
METHODS—100 patients underwent implantation of a hydrogel IOL in one eye and an acrylic IOL implantation in the opposite eye. The area of the anterior capsule opening, and the degree of IOL decentration and tilt were measured using the Scheimpflug videophotography system at 3 days, and at 1, 3, and 6 months postoperatively.
RESULTS—The mean anterior capsule opening area decreased significantly in both groups. At 6 months postoperatively, the area in the hydrogel group was significantly smaller than that in the acrylic group. The mean percentage of the area reduction in the hydrogel group was also significantly greater than that in the acrylic group, being 16.9% in the hydrogel group and 8.8% in the acrylic group. In contrast, IOL decentration and tilt did not progress in either group. No significant differences were found in the degree of IOL decentration and tilt throughout the follow up period.
CONCLUSIONS—Contraction of the anterior capsule opening was more extensive with the hydrogel IOL than with the acrylic IOL, but the degree of IOL decentration and tilt were similar for the two types of lenses studied.

PMCID: PMC1723752  PMID: 11673291
2.  Anterior capsule contraction and intraocular lens dislocation in eyes with pseudoexfoliation syndrome 
The British Journal of Ophthalmology  1998;82(12):1429-1432.
AIMS—To examine the extent of anterior capsule contraction as well as intraocular lens (IOL) decentration and tilt following implant surgery in eyes with pseudoexfoliation syndrome (PE).
METHODS—53 eyes from 53 patients with PE and 53 control eyes from 53 age matched patients, undergoing phacoemulsification and implant surgery, were recruited. The anterior capsule opening area and the amounts of IOL decentration and tilt after undergoing continuous curvilinear capsulorhexis were measured using the Scheimpflug videophotography system at 1 week and 1, 3, 6, 9, and 12 months postoperatively.
RESULTS—The mean area of the anterior capsule opening in the PE group was significantly smaller than that in the control group at 1 month postoperatively and later. The percentage reductions in the PE group were approximately 25%, while they were less than 10% in the control group. The degree of IOL tilt was also larger in the PE group than in the control group. Five eyes (9.4%) in the PE group underwent a neodymium:YAG laser anterior capsulotomy, but none in the control group underwent a capsulotomy.
CONCLUSIONS—The contraction of the anterior capsule opening was more extensive in the PE eyes than in the control eyes, thus resulting in a high Nd:YAG laser anterior capsulotomy rate. The IOL tilt was also greater in the PE eyes than in the control eyes.

 Keywords: anterior capsule contraction; intraocular lens dislocation; pseudoexfoliation syndrome; continuous curvilinear capsulorhexis
PMCID: PMC1722461  PMID: 9930277
3.  Comparison of decentration and tilt between one piece and three piece polymethyl methacrylate intraocular lenses 
BACKGROUND—The extent of the decentration and tilt was prospectively compared between one piece polymethyl methacrylate (PMMA) and three piece PMMA intraocular lenses (IOLs) which were implanted in the capsular bag after performing continuous curvilinear capsulorhexis.
METHODS—91 patients underwent a one piece PMMA IOL implantation in one eye as well as the implantation of the three piece PMMA IOL with polyvinylidene fluoride loops in the opposite eye. The length of the lens decentration and the angle of the tilt were quantitated using the anterior eye segment analysis system (EAS-1000) at 1 week as well as 1, 3, and 6 months postoperatively.
RESULTS—The mean length of the decentration in the one piece IOL was smaller than that in the three piece IOL at 1 week (p=0.0092), 1 month (p=0.0044), 3 months (p=0.0069), and 6 months (p=0.0010) postoperatively. However, no significant difference was found in the degree of the tilt between the two types of IOLs throughout the observation periods.
CONCLUSION—These results clarified that the one piece PMMA IOL with rigid PMMA haptics implanted in the capsular bag provides a better centration than the three piece PMMA IOL with flexible haptics, whereas the tilt was the same between the two types of IOLs.

 Keywords: intraocular lens; decentration; tilt; continuous curvilinear capsulorhexis
PMCID: PMC1722563  PMID: 9640193
4.  Effect of methylglyoxal on multidrug-resistant Pseudomonas aeruginosa 
Honey has a complex chemistry, and its broad-spectrum antimicrobial activity varies with floral source, climate, and harvesting conditions. Methylglyoxal was identified as the dominant antibacterial component of manuka honey. Although it has been known that methylglyoxal has antibacterial activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, there is not much information describing its activity against gram-negative bacteria. In this study, we report the effect of methylglyoxal against multidrug-resistant Pseudomonas aeruginosa (MDRP) using 53 clinically isolated strains. We also assessed the effect of deleting the five multidrug efflux systems in P. aeruginosa, as well as the efflux systems in Escherichia coli and Salmonella enterica serovar Typhimurium, on MICs of methylglyoxal. Our results indicate that methylglyoxal inhibits the growth of MDRP at concentrations of 128–512 μg/ml (1.7–7.1 mM) and is not recognized by drug efflux systems.
PMCID: PMC4029012  PMID: 24860556
manuka honey; methylglyoxal; drug efflux system; multidrug resistance; Pseudomonas aeruginosa
5.  Risk factors for an early dialysis start in patients with diabetic nephropathy end-stage renal disease 
Patients with end-stage renal disease (ESRD) have symptoms related to severe anemia, edema, and heart failure. Although dialysis improves ESRD syndromes, the optimum timing for initiation of dialysis is unclear. Recent observational studies have suggested that early commencement of dialysis can be harmful. Given that early dialysis may increase the risk of death, avoiding an early start to dialysis is recommended. Patients with diabetic nephropathy (DN) may have risk factors for early dialysis. However, the risk factors for early dialysis are unclear in ESRD patients with DN. The aim of this study was to elucidate the risk factors for early initiation of dialysis in patients with DN and ESRD.
From April 2009 to December 2012, we identified Japanese DN patients with an estimated glomerular filtration rate of less than 15 mL/minute/1.73 m2. The patients were divided into late or early dialysis groups based on the timing of start of dialysis.
We evaluated 52 patients who commenced dialysis during the observation period, including 33 in the late dialysis group and 19 in the early dialysis group. There was a significant association between early dialysis and age ≥65 years (odds ratio 4.59). The incidence of pneumonia before starting dialysis was significantly higher in elderly patients than in nonelderly patients.
Our findings suggest that elderly patients with DN and ESRD have an increased risk of early initiation of dialysis, and occurrence of pneumonia is also associated with early dialysis. To avoid early commencement of dialysis, booster pneumococcal vaccination could be useful in elderly DN patients with ESRD.
PMCID: PMC3913608  PMID: 24511237
early dialysis; diabetes mellitus; nephropathy; elderly patients; estimated glomerular filtration rate
6.  Restructuring the Ikeda City school urinary screening system: report of a screening survey 
Annual urinary screening is conducted at municipal kindergartens, elementary schools, and junior high schools in Ikeda City, Osaka, Japan (Ikeda City School System), and the results are reviewed by a general physician, but standards for when to recommend specialist referral have not been clear.
In all children attending the Ikeda City School System in 2012, dipstick urinalysis of a first-morning urine specimen was recommended once or twice, and if a second urinalysis showed proteinuria (≥1+), the urinary protein/creatinine ratio was measured. If this showed ≥0.2 g/g of creatinine (g/gCr), it was recommended that the child be evaluated by a specialist at Ikeda City Hospital.
Urinary screening was performed in about 20% (388) of kindergarten, about 90% (5363) of elementary school, and about 86% (2523) of junior high school children living in Ikeda City. Urine samples were obtained from 387, 5349, and 2476 children, respectively. The urinary protein/creatinine ratio was ≥0.2 g/gCr in 13 children, including 1 elementary and 12 junior high children. In these 13 children, chronic nephritic syndrome (CNS) was suspected in 6 junior high school children, and of these, this was a new finding in 5, and renal biopsy was indicated in 3. In Ikeda City, the prevalence of CNS in elementary school children was <0.03%, the prevalence of CNS in junior high school children was 0.29%, and a renal biopsy was indicated in 0.14%. By eliminating the costs associated with assessment of the results by the Ikeda Medical Association, and by directly contracting with the testing company, the expenses paid by Ikeda City for the system itself decreased from 2,508,619 yen to 966,157 yen.
Incorporating the urinary protein/creatinine ratio into the school urinary screening system in the Ikeda City School System and clarifying standards for specialist referral has enabled restructuring of the system so that is efficient and its effectiveness can be assessed.
PMCID: PMC4028752  PMID: 24330222
Chronic glomerulonephritis; Chronic nephritic syndrome; Prevalence; Renal biopsy; School urinary screening; Urinary protein/creatinine ratio
7.  A New Protocol to Detect Multiple Foodborne Pathogens with PCR Dipstick DNA Chromatography after a Six-Hour Enrichment Culture in a Broad-Range Food Pathogen Enrichment Broth 
BioMed Research International  2013;2013:295050.
A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5–10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments.
PMCID: PMC3865639  PMID: 24364031
8.  Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages 
Cell Death and Differentiation  2012;19(11):1856-1869.
Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.
PMCID: PMC3469054  PMID: 22790873
Bcl-xL; Mcl-1; apoptosis; megakaryocyte; platelet; reticulated platelet
9.  Sigma-1 Receptor Chaperone at the ER-Mitochondrion Interface Mediates the Mitochondrion-ER-Nucleus Signaling for Cellular Survival 
PLoS ONE  2013;8(10):e76941.
The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.
PMCID: PMC3799859  PMID: 24204710
10.  Regression of cerebellar tonsillar descent and hydrocephalus after endoscopic third ventriculostomy in a patient with a quadrigeminal arachnoid cyst 
Posterior fossa arachnoid cysts, including quadrigeminal cistern arachnoid cysts, can occasionally cause compression of the quadrigeminal plate, leading to Sylvian aqueduct stenosis and induction of cerebellar tonsillar descent into the foramen magnum. This, in turn, can result in obstructive hydrocephalus. In such cases, the characteristic of hydrocephalus is generally considered to be hypertensive.
Case Description:
We present the case of a 28-year-old female complaining of chronic and progressively worsening headaches following the delivery of her first child. Magnetic resonance imaging revealed marked tri-ventriculomegaly, the arachnoid cyst located in the quadrigeminal cistern, and cerebellar tonsillar descent. Ophthalmoscopy revealed bilateral papilledema indicating a long-standing elevation of intracranial pressure. Endoscopic third ventriculostomy (ETV) was performed successfully and resulted in complete recovery from her headaches and papilledema. Postoperative MRI revealed resolution of ventriculomegaly and cerebellar tonsillar descent, suggesting that the fourth ventricle outlet obstruction was associated with the development of the hydrocephalus in this patient.
Our case is the first report that a quadrigeminal arachnoid cyst associated with both cerebellar tonsillar descent and hydrocephalus was well treated with ETV. It was indicated that the patient's hydrocephalus and cerebellar tonsillar descent were secondary and synergistic events, caused by the arachnoid cyst located in the quadrigeminal cistern.
PMCID: PMC3815032  PMID: 24232572
Arachnoid cyst; endoscopic third ventriculostomy; hydrocephalus; tonsillar descent
11.  Thallium group poisoning incident in Japan 2011 
Critical Care  2013;17(Suppl 2):P269.
PMCID: PMC3642765
12.  Orbital Cystic Schwannoma Originating from the Frontal Nerve 
Schwannomas of the orbit are very rare benign neoplasms. Intraorbital cystic schwannomas originating from the frontal nerve are even rarer, with only 1 case reported to date. This is most likely due to the fact that, in most cases, the origin of the orbital schwannoma cannot be identified intraoperatively. The nerve origin is usually speculated from histological examination of the specimen and the postoperative neurological deficits of the patient. Here, we present the case of a 65-year-old woman with a one-month history of exophthalmos, whose orbital cystic lesion was completely removed by microsurgical transcranial operation. Intraoperatively, the continuity between the tumor and frontal nerve was seen macroscopically, leading us to confirm the frontal nerve as an origin of the tumor, which was consistent with the postoperative neurological findings. The diagnosis of the tumor was established as schwannoma from the histological examination. As a differential diagnosis of the orbital cystic lesions, the possibility of schwannomas should be kept in mind.
PMCID: PMC3540697  PMID: 23320224
13.  -449 C>G polymorphism of NFKB1 gene, coding nuclear factor-kappa-B, is associated with the susceptibility to ulcerative colitis 
AIM: To clarify the association between a polymorphism -449 C>G (rs72696119) in 5’-UTR of NFKB1 with ulcerative colitis (UC).
METHODS: The studied population comprised 639 subjects, including patients with UC (UC cases, n = 174) and subjects without UC (controls, n = 465). We employed polymerase chain reaction-single strand conformation polymorphism to detect the gene polymorphism.
RESULTS: The rs72696119 G allele frequencies in controls and UC cases were 33.4% and 38.5%, respectively (P = 0.10). Genotype frequency of the GG homozygote in UC cases was significantly higher than that in controls (P = 0.017), and the GG homozygote was significantly associated with susceptibility to UC [odds ratio (OR), 1.88; 95%CI, 1.13-3.14]. In male subjects, the GG homozygote was associated with an increased risk for UC (OR, 3.10; 95%CI, 1.47-6.54; P = 0.0053), whereas this association was not found in female subjects. In addition, the GG homozygote was significantly associated with the risk of non-continuous disease (OR, 2.06; 95%CI, 1.12-3.79; P = 0.029), not having total colitis (OR, 2.40; 95%CI, 1.09-3.80, P = 0.040), disease which developed before 20 years of age (OR, 2.80; 95%CI, 1.07-7.32, P = 0.041), no hospitalization (OR, 2.28; 95%CI, 1.29-4.05; P = 0.0090) and with a maximum of 8 or less on the UCDAI score (OR, 2.45; 95%CI, 1.23-4.93; P = 0.022).
CONCLUSION: Our results provide evidence that NFKB1 polymorphism rs72696119 was significantly associated with the development of UC. This polymorphism influences the susceptibility to and pathophysiological features of UC.
PMCID: PMC3531683  PMID: 23322997
Genetic polymorphism; NFKB1; Ulcerative colitis
14.  Role of Histamine and Its Receptor Subtypes in Stimulation of Conjunctival Goblet Cell Secretion 
The purpose of this study was to determine the effect of histamine and its receptors on goblet cell secretion.
Cultured rat and human goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugate was measured by an enzyme-linked lectin assay. Cultured rat goblet cells were homogenized and either RNA was isolated for RT-PCR or proteins were isolated for Western blot analysis for presence of histamine receptors subtypes H1 through H4. The localization of these receptors was determined in rat and human goblet cells by immunofluorescence microscopy.
Histamine stimulated goblet cell secretion in a concentration- and time-dependent manner. All four histamine receptors were present in cultured rat and human goblet cells. Use of agonists specific to individual histamine receptor subtypes indicated that the rank order of agonist stimulation was H1 = H3 > H4 > H2. Using antagonists specific to individual histamine receptor subtypes determined that H2 and H3, but not the H1 and H4, antagonists, inhibited histamine-stimulated conjunctival goblet cell secretion.
Rat and human conjunctival goblet cells are a direct target of histamine, which induces secretion. All four histamine receptors are present in rat and human conjunctiva and are active in rat conjunctival goblet cells. These findings suggest that all four histamine receptor subtypes are important for conjunctival goblet cell secretion. Blockage of histamine receptor subtypes could prevent the excess mucus production associated with ocular allergy.
The study demonstrated that all four histamine receptors are present and active in rat conjunctival goblet cells.
PMCID: PMC3376076  PMID: 22467574
15.  Dienogest increases the progesterone receptor isoform B/A ratio in patients with ovarian endometriosis 
The resistance of endometriotic tissue to progesterone can be explained by alterations in the distribution of progesterone receptor (PR) and estrogen receptor (ER) isoforms. The aims of this study were to examine the expressions of PR-A, PR-B, ERα and ERβ in endometrioma and assess whether these expressions are affected by dienogest or leuprolide acetate (LA) treatment.
We enrolled 60 females, including 43 patients with endometriosis (14 who received no medical treatment, 13 who received dienogest and 16 who received LA before undergoing laparoscopic surgery) and 17 patients with leiomyoma. The expression levels of PR and ER isoforms in eutopic and ectopic endometrium were assayed with quantitative real-time PCR, and confirmed with immunohistochemistry.
A decreased PR-B/PR-A ratio and an increased ERβ/ERα ratio were demonstrated in ectopic endometrium derived from females with endometriosis compared with the ratios observed in eutopic endometrium obtained from females without endometriosis. Although LA treatment did not affect the PR-B/PR-A and ERβ/ERα ratios, dienogest treatment increased the PR-B/PR-A ratio and decreased the ERβ/ERα ratio in patients with endometriomas.
Dienogest may improve progesterone resistance in endometriotic tissue by increasing the relative expressions of PR-B and PR-A, and decreasing the relative expressions of ERβ and ERα.
PMCID: PMC3541078  PMID: 23113924
Dienogest; Progesterone receptor isoforms; Estrogen receptor isoforms; Ovarian endometriosis; Progesterone resistance
16.  Inhibitory effects of carbon dioxide insufflation on pneumoperitoneum and bowel distension after percutaneous endoscopic gastrostomy 
AIM: To evaluate the inhibitory effects of carbon dioxide (CO2) insufflation on pneumoperitoneum and bowel distension after percutaneous endoscopic gastrostomy (PEG).
METHODS: A total of 73 consecutive patients who were undergoing PEG were enrolled in our study. After eliminating 13 patients who fitted our exclusion criteria, 60 patients were randomly assigned to either CO2 (30 patients) or air insufflation (30 patients) groups. PEG was performed by pull-through technique after three-point fixation of the gastric wall to the abdominal wall using a gastropexy device. Arterial blood gas analysis was performed immediately before and after the procedure. Abdominal X-ray was performed at 10 min and at 24 h after PEG to assess the extent of bowel distension. Abdominal computed tomography was performed at 24 h after the procedure to detect the presence of pneumoperitoneum. The outcomes of PEG for 7 d post-procedure were also investigated.
RESULTS: Among 30 patients each for the air and the CO2 groups, PEG could not be conducted in 2 patients of the CO2 group, thus they were excluded. Analyses of the remaining 58 patients showed that the patients’ backgrounds were not significantly different between the two groups. The elevation values of arterial partial pressure of CO2 in the air group and the CO2 group were 2.67 mmHg and 3.32 mmHg, respectively (P = 0.408). The evaluation of bowel distension on abdominal X ray revealed a significant decrease of small bowel distension in the CO2 group compared to the air group (P < 0.001) at 10 min and 24 h after PEG, whereas there was no significant difference in large bowel distension between the two groups. Pneumoperitoneum was observed only in the air group but not in the CO2 group (P = 0.003). There were no obvious differences in the laboratory data and clinical outcomes after PEG between the two groups.
CONCLUSION: There was no adverse event associated with CO2 insufflation. CO2 insufflation is considered to be safer and more comfortable for PEG patients because of the lower incidence of pneumoperitoneum and less distension of the small bowel.
PMCID: PMC3400858  PMID: 22826621
Percutaneous endoscopic gastrostomy; Carbon dioxide insufflation; Pneumoperitoneum; Abdominal distension; Randomized control study
17.  Salvage radiotherapy after radical prostatectomy: outcomes and prognostic factors especially focusing on pathological findings 
Journal of Radiation Research  2012;53(5):727-734.
External beam radiotherapy is a potential salvage or adjuvant therapy after radical prostatectomy (RP). The purpose of this study was to investigate the treatment outcome of salvage radiotherapy (RT) following RP for clinically localized prostate cancer and to identify factors that may predict the outcome of salvage RT. Between 2000 and 2006, 41 patients received salvage RT because of increasing prostate-specific antigen (PSA) levels following an RP for clinically localized prostate cancer. All the patients received conformal radiotherapy to the prostate bed. The prescribed radiation dose was 60–70 Gy in 26–35 fractions. The overall 5-year biochemical disease-free survival rate was 38%. A multivariate analysis showed that the following pathological findings of the surgical specimen were significantly associated with biochemical failure following salvage RT: a high Gleason score, a negative surgical margin, seminal vesicle invasion, lymphatic vessel invasion and negative vascular invasion. Among these factors, lymphatic vessel invasion was the strongest predictor. In conclusion, the pathological features affected the outcome of salvage RT following RP. Lymphatic vessel invasion was strongly associated with the risk of biochemical failure despite salvage RT. Meanwhile, vascular invasion was not a significant hazardous factor.
PMCID: PMC3430423  PMID: 22843370
prostatectomy; salvage radiotherapy; prostate cancer; prostate-specific antigen; pathological findings
18.  Antigen Retrieval to Improve the Immunocytochemistry Detection of Sigma-1 Receptors and ER Chaperones 
Histochemistry and cell biology  2011;135(6):627-637.
Molecular chaperones localized at the endoplasmic reticulum (ER) lumen constitutively or cellular stress-dependently associate with a variety of proteins to promote their proper folding or to inhibit protein misfolding. ER chaperones preferentially form large complexes with co-chaperones and/or misfolded proteins in a highly crowded cellular environment that often hampers their detection by immunocytochemistry (ICC). This study establishes an antigen retrieval (AR) protocol to improve the ICC detection of ER chaperones in cultured cells using widely available antibodies against synthetic peptides. Among ten different antigen retrieval/fixation conditions, only the AR with Tris-HCl (pH 9.5) containing 6 M urea (80 °C for 10 min) significantly improved the ICC detection of the novel ER chaperone sigma-1 receptor (Sig-1R) in Chinese hamster ovary cells. Extended fixation with 4% paraformaldehyde for 1 hr effectively preserved the morphology of the ER under the AR condition. This method greatly enhanced the signal-to-noise ratio in Sig-1R ICC, thus allowing for semi-quantitative detection of protein upregulation under ER stress. The AR similarly improved the ICC detection of a series of other major ER chaperones, including BiP/GRP78, GRP94, calnexin, calreticulin, ERp57, protein disulfide isomerase, and cyclophilin B. The improved ICC methodology using the urea AR at 80°C may improve ICC of ER molecules as well as visualization of ER structure and substructures.
PMCID: PMC3155709  PMID: 21573736
antigen retrieval; urea; molecular chaperone; endoplasmic reticulum; sigma-1 receptor; ER stress
19.  MPO-ANCA associated crescentic glomerulonephritis with numerous immune complexes: case report 
BMC Nephrology  2012;13:32.
Antineutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis (CGN) is a major cause of rapidly progressive glomerulonephritis (RPGN). ANCA-associated CGN is generally classified into pauci-immune RPGN, in which there are few or no immune complexes.
Case Presentation
A 78-year-old man presented with RPGN after a 7-year course of chronic proteinuria and hematuria with stable renal function. A blood examination showed a high titer of myeloperoxidase (MPO)-ANCA. A renal biopsy showed crescentic glomerulonephritis with abundant subepithelial, intramenbranous and subendothelial deposits by electron microscopy, leading to the diagnosis of ANCA-associated CGN superimposed on type 3 membranoproliferative glomerulonephritis (MPGN).
This case is unique in that type 3 MPGN and MPO-ANCA-associated CGN coexisted, and no similar case has been reported to date. Because ANCA-associated CGN has a predilection for elderly individuals and primary type 3 MPGN is rarely seen in this age group, coincidental existence appears less likely. This case may confer valuable information regarding the link between immune complex and ANCA-associated CGN.
PMCID: PMC3470990  PMID: 22656245
Myeloperoxidase-antineutrophil cytoplasmic antibody; Membranoproliferative glomerulonephritis; Immune complex; Rapidly progressive glomerulonephritis
20.  Synthesis and Characterization of PEGylated Toll Like Receptor 7 Ligands 
Bioconjugate chemistry  2011;22(3):445-454.
Toll like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid or polyethylene glycol (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6–10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1β and type 1 interferon, as well for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands.
PMCID: PMC3063487  PMID: 21338093
polyethylene glycol; PEG; Toll like receptor 7
21.  Monoallelic Expression of Krt12 Gene during Corneal-type Epithelium Differentiation of Limbal Stem Cells 
Expression of the keratin 12 (Krt12) gene is subjected to monoallelic regulation, as demonstrated by the use of bitransgenic Krt12cre/+/ROSAEGFP mice. This allelic selection of Krt12 gene expression is advantageous for retaining epithelial cells expressing the Krt12+ allele and allows tolerance to structural mutations of Krt12 in persons carrying heterozygous mutations.
The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the keratin 12 (Krt12) gene during corneal type-epithelium differentiation.
Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSAEGFP, Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12Cre/+/ROSAEGFP mice were examined by fluorescence-activated cell sorter.
Mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12Cre/+/ZEG mice, respectively. Immunostaining revealed that Cre− cells were also Krt12 negative in the corneal epithelia of Krt12Cre/−/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Cre/+/ROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP− cells express only Krt12+. In the Krt12Cre/− cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Cre/Cre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed.
These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.
PMCID: PMC2941181  PMID: 20393120
22.  Detection of Hepatitis B and C Viruses in Almost All Hepatocytes by Modified PCR-Based In Situ Hybridization▿  
Journal of Clinical Microbiology  2010;48(11):3843-3851.
Although PCR-based in situ hybridization (PCR-ISH) can be used to determine the distribution and localization of pathogens in tissues, this approach is hampered by its low specificity. Therefore, we used a highly specific and sensitive PCR-ISH method to reveal the lobular distribution and intracellular localization of hepatitis B virus (HBV) and HCV in chronic liver disease and to clarify the state of persistent HBV and HCV infection in the liver. HBV genomic DNA was detected in almost all hepatocytes, whereas HBV RNA or protein was differentially distributed only in a subset of the HBV DNA-positive region. Further, HCV genomic RNA was detected in almost all hepatocytes and was localized to the cytoplasm. HCV RNA was also detected in the epithelium of the large bile duct but not in endothelial cells, portal tracts, or sinusoidal lymphocytes. In patients with HBV and HCV coinfection, HCV RNA was localized to the noncancerous tissue, whereas HBV DNA was found only in the cancerous tissue. Using this novel PCR-ISH method, we could visualize the staining pattern of HBV and HCV in liver sections, and we obtained results consistent with those of real-time detection (RTD)-PCR analysis. In conclusion, almost all hepatocytes are infected with HBV or HCV in chronic liver disease; this finding implies that the viruses spreads throughout the liver in the chronic stage.
PMCID: PMC3020860  PMID: 20739486
23.  A case of intrahepatic clear cell cholangiocarcinoma 
Intrahepatic clear cell cholangiocarcinoma is very rare - only 8 cases have been reported. A 56-year-old Japanese man with chronic hepatitis B infection was diagnosed with a 2.2 cm hepatocellular carcinoma on imaging, and hepatic segmentectomy was performed. Histopathologically, the tumor cells had copious clear cytoplasm and formed glandular structures or solid nests. These pathological findings suggested the tumor was a clear cell variant of intrahepatic cholangiocarcinoma. Particular stains and radiological images suggested that the cause of the clear cell change had been glycogen, not mucin nor lipid. On immunohistochemical staining, cytokeratin (CK) 7 and CK19 were positive, whereas CK20 was negative. Vimentin was detected on the cell membranes, and CD56 was focally positive. The patient was given adjuvant chemotherapy and is currently free from the tumor 7 mo postoperatively. Careful follow-up with adequate postoperative supplementary chemotherapy is necessary because the characteristics of this type of tumor are unknown.
PMCID: PMC2877190  PMID: 20503460
Intrahepatic cholangiocarcinoma; Clear cell carcinoma; Glycogen; Mucin; Lipid; Hepatectomy
24.  Excess biglycan causes eyelid malformation by perturbing muscle development and TGF-α signaling 
Developmental biology  2005;277(1):222-234.
Tissue morphogenesis during development is regulated by growth factors and cytokines, and is characterized by constant remodeling of extracellular matrix (ECM) in response to signaling molecules, for example, growth factors, cytokines, and so forth. Proteoglycans that bind growth factors are potential regulators of tissue morphogenesis during embryonic development. In this study, we showed that transgenic mice overexpressing biglycan under the keratocan promoter exhibited exposure keratitis and premature eye opening from noninfectious eyelid ulceration due to perturbation of eyelid muscle formation and the failure of meibomian gland formation. In addition, in vitro analysis revealed that biglycan binds to TGF-α, thus interrupting EGFR signaling pathways essential for mesenchymal cell migration induced by eyelid epithelium. The defects of TGF-α signaling by excess biglycan were further augmented by the interruption of the autocrine or paracrine loop of the EGFR signaling pathway of HB-EGF expression elicited by TGF-α. These results are consistent with the notion that under physiological conditions, biglycan secreted by mesenchymal cells serves as a regulatory molecule for the formation of a TGF-α gradient serving as a morphogen of eyelid morphogenesis.
PMCID: PMC2876305  PMID: 15572151
Biglycan; Eyelid morphogenesis; TGF-α; Muscle development; Keratocan promoter; Transgenic mouse
25.  The postsynaptic density proteins Homer and Shank form a polymeric network structure 
Cell  2009;137(1):159-171.
The postsynaptic density (PSD) is crucial for synaptic functions, but the molecular architecture retaining its structure and components remains elusive. Homer and Shank are among the most abundant scaffolding proteins in the PSD, working synergistically for maturation of dendritic spines. Here, we demonstrate that Homer and Shank, together, form a mesh-like matrix structure. Crystallographic analysis of this region revealed a pair of parallel dimeric coiled-coils intercalated in a tail-to-tail fashion to form a tetramer, giving rise to the unique configuration of a pair of amino-terminal EVH1 domains at each end of the coiled-coil. In neurons, the tetramerization is required for structural integrity of the dendritic spines and recruitment of proteins to synapses. We propose that the Homer-Shank complex serves as a structural framework and as an assembly platform for other PSD proteins.
PMCID: PMC2680917  PMID: 19345194
Postsynaptic density; Homer; Shank; crystal structure; coiled-coil; synapse; scaffolding protein

Results 1-25 (2018)