Mayumi Sakurai

Differential recognition of heat shock elements by members of the heat shock transcription factor family

Heat shock transcription factor (HSF), an evolutionarily conserved stress response regulator, forms trimers and binds to heat shock element (HSE), comprising at least three continuous inverted repeats of the sequence 5′‐nGAAn‐3′. The single HSF of yeast is also able to bind discontinuously arranged nGAAn units. We investigated interactions between three human HSFs and various HSE types in vitro, in yeast cells, and in HeLa cells. Human HSF1, a stress‐activated regulator, preferentially bound to continuous HSEs rather than discontinuous HSEs, and heat shock of HeLa cells caused expression of reporter genes containing continuous HSEs. HSF2, whose function is implicated in neuronal specification and spermatogenesis, exhibited a slightly higher binding affinity to discontinuous HSEs than did HSF1. HSF4, a protein required for ocular lens development, efficiently recognized discontinuous HSEs in a trimerization‐dependent manner. Among four human γ‐crystallin genes encoding structural proteins of the lens, heat‐induced HSF1 preferred HSEs on the γA‐crystallin and γB‐crystallin promoters, whereas HSF4 preferred HSE on the γC‐crystallin promoter. These results suggest that the HSE architecture is an important determinant of which HSF members regulate genes in diverse cellular processes.

Isolating triamcinolone acetonide particles for intravitreal use with a porous membrane filter.

Purpose To report a new, simple, rapid method to isolate triamcinolone acetonide particles and to remove additives from its commercially available suspension (Kenacort-A) for intravitreal use. Methods The contents of a Kenacort-A vial (40 mg triamcinolone acetonide suspended in 1.0 mL vehicle) were loaded into a syringe and passed through a porous membrane filter with 0.45-&mgr;m pores. The filter was then backflushed with distilled water to yield a vehicle-poor suspension of triamcinolone acetonide in the initial syringe. This filtration and backflush procedure was repeated four times, and each waste filtrate was subjected to high-performance liquid chromatography to identify benzyl alcohol, a preservative in the vehicle. Gel permeation chromatography was also used to determine the degree to which carboxymethylcellulose, one of the two suspending agents in the vehicle, permeated the membrane filter. Although 7.5 mg/mL high-viscosity carboxymethylcellulose hardly passed through the 0.45-&mgr;m pore filter, it passed through the 5.0-&mgr;m pore filter easily. Therefore, a 5.0-&mgr;m pore filter was used in this study. Results By using a 0.45-&mgr;m porous membrane filter, 99.7% of the benzyl alcohol can be eliminated. By using a 5.0-&mgr;m porous membrane filter, but not by using a 0.45-&mgr;m porous membrane filter, 88.1% of the high-viscosity carboxymethylcellulose can be eliminated. Conclusions The filtration and backflush procedure using the 5.0-&mgr;m porous membrane filters is useful during vitrectomy to reduce the preparation time of triamcinolone acetonide suspension. Also, this method of reducing additives may be more helpful when using triamcinolone as a therapeutic agent for intravitreal depot use, because there is no washout effect when it is used in this manner.

Analysis of COL8A2 Gene Mutation in Japanese Patients with Fuchs’ Endothelial Dystrophy and Posterior Polymorphous Dystrophy

PurposeTo determine whether Japanese patients with Fuchs’ endothelial corneal dystrophy (FECD) and posterior polymorphous dystrophy (PPMD) carry mutations in the COL8A2 gene, and to investigate the possible pathogenicity of the COL8A2 gene in these corneal dystrophies.MethodsDNA analysis of the COL8A2 gene was performed in 15 unrelated Japanese patients with FECD, and 5 patients with PPMD using polymerase chain reaction and direct sequencing. Mutation screenings were also performed in 36 unrelated normal volunteers as controls, as well as slit-lamp and specular microscopy.ResultsTwo types of heterozygous missense mutations of the COL8A2 gene (R155Q and T502M) in 5 of 15 FECD probands (R155Q, 3/30 chromosomes, 10.0%; T502M, 3/30 chromosomes, 10.0%) were found. No mutation was detected in the coding region of the COL8A2 gene in the remaining 10 patients with FECD nor in any of the 5 patients with PPMD. These two mutations were also found in normal Japanese volunteers (R155Q, 5/72 chromosomes, 6.9%; T502M, 11/70 chromosomes, 15.7%). The chromosomal frequency of the two mutations was not significant between the patients and normal controls.ConclusionsThe R155Q and T502M mutations of COL8A2 may not be the causative defect in the Japanese FECD and PPMD patients examined in this study. Jpn J Ophthalmol 2004;48:195–198

Endophthalmitis Caused by Enterococcus mundtii

ABSTRACT Enterococcus mundtii has rarely been isolated from environmental or human sources. We report the identification of E. mundtii as a pathogen of human infectious disease by DNA sequencing of 16S rRNA and sodA genes in a case of endophthalmitis developed in a 66-year-old immunocompetent gardener.

Discrepancy of the intraocular pressure response between fellow eyes in one-eye trials versus bilateral treatment: verification with normal subjects.

PurposeTo compare the correlation of the fellow-eyes intraocular pressure (IOP) response in one-eye trials performed separately for each eye with that of bilateral treatment in normal subjects. MethodsA one-eye trial with topical latanoprost applied once daily for 7 days was carried out in the right eye and then in the left eye of 41 normal subjects. Bilateral treatment was performed in a different set of 41 normal subjects. IOPs were measured at 3 time points on day 0 and on day 7. ResultsLatanoprost significantly reduced IOP of treated eyes in one-eye trials (2.8±1.6 and 2.7±1.6 mm Hg in the first and second trial, respectively) and in bilateral treatments (2.8±1.3 and 2.6±1.4 mm Hg in the right and left eye, respectively). Correlation of mean diurnal IOP reduction between 2 one-eye trials was poor (r2=0.102), even after subtracting the nontreated eye IOP fluctuations from the treated eye IOPs (r2=0.097), but that between fellow eyes in bilateral treatment was excellent (r2=0.849). Correlation of baseline IOP at each time point between fellow eyes in one-eye trials and bilateral treatment (r2=0.729 to 0.949) was better than that in the same eye between 2 one-eye trials (r2=0.319 to 0.631). ConclusionsFellow eyes in normal subjects showed a symmetrical IOP response to short-term bilateral treatment with latanoprost, although they did not respond symmetrically to one-eye trials performed separately for each eye. Poor correlation of IOP changes between 2 one-eye trials may be caused by different IOP responsiveness to latanoprost at each trial, rather than asymmetrical IOP fluctuations.

Association between genetic polymorphisms of the prostaglandin F2α receptor gene, and response to latanoprost in patients with glaucoma and ocular hypertension

Aim To examine whether intraocular pressure (IOP) reduction by latanoprost correlates with single nucleotide polymorphisms (SNPs) of the prostaglandin F2α (FP) receptor gene in patients with glaucoma and ocular hypertension (OH). Methods The genotype of nine SNPs in the FP receptor gene was determined by direct DNA sequencing, or other techniques, in 82 patients with glaucoma or OH who were treated with latanoprost monotherapy in one eye. The IOP reduction was evaluated by the percent IOP reduction (%ΔIOP), estimated by subtracting IOP fluctuations in the untreated fellow eye. Subjects were classified by %ΔIOP into low responders (%ΔIOP<10%) and others (%ΔIOP ≥10%). The correlation between %ΔIOP and SNPs in the FP receptor gene was analysed. Results Multiple regression analysis demonstrated that the rs12093097 was the only significant factor that correlated with %ΔIOP (p=0.039). Among estimated haplotypes, one haplotype that contained the minor allele only in rs3753380, was significantly correlated with low responders even after correction for multiple test (permutation test, p=0.037). Conclusions An association was found between SNPs of the FP receptor gene and the response to latanoprost in patients with glaucoma or OH. The FP receptor genetic polymorphism may influence the degree of IOP reduction by latanoprost in these patients.

Association of PAX2 and Other Gene Mutations with the Clinical Manifestations of Renal Coloboma Syndrome

Background Renal coloboma syndrome (RCS) is characterized by renal anomalies and optic nerve colobomas. PAX2 mutations contribute to RCS. However, approximately half of the patients with RCS have no mutation in PAX2 gene. Methods To investigate the incidence and effects of mutations of PAX2 and 25 candidate genes, patient genes were screened using next-generation sequence analysis, and candidate mutations were confirmed using Sanger sequencing. The correlation between mutations and clinical manifestation was evaluated. Result Thirty patients, including 26 patients (two families of five and two, 19 sporadic cases) with RCS, and 4 optic nerve coloboma only control cases were evaluated in the present study. Six PAX2 mutations in 21 probands [28%; two in family cohorts (n = 5 and n = 2) and in 4 out of 19 patients with sporadic disease] including four novel mutations were confirmed using Sanger sequencing. Moreover, four other sequence variants (CHD7, SALL4, KIF26B, and SIX4) were also confirmed, including a potentially pathogenic novel KIF26B mutation. Kidney function and proteinuria were more severe in patients with PAX2 mutations than in those without the mutation. Moreover, the coloboma score was significantly higher in patients with PAX2 gene mutations. Three out of five patients with PAX2 mutations had focal segmental glomerulosclerosis (FSGS) diagnosed from kidney biopsies. Conclusion The results of this study identify several new mutations of PAX2, and sequence variants in four additional genes, including a novel potentially pathogenic mutation in KIF26B, which may play a role in the pathogenesis of RCS.

Characterization and diabetes-induced impairment of nitric oxide synthase in rat choroid

Purpose. To examine the nitric oxide (NO) system in the choroid of normal rats and rats with streptozotocin (STZ)-induced diabetes. Methods. We assayed NO synthase (NOS) activity by monitoring the conversion of L -[ 14 C] arginine to L -[ 14 C] citrulline, identified the NOS isoforms by immunoblotting, and examined the effects of STZ-induced diabetes on NOS in the rat choroid. Results. Calcium-independent NOS activity was insignificant in the choroid of normal and diabetic rats. Choroidal calcium-dependent NOS activity was high and comparable to that in the cerebellum. Neuronal (n) NOS protein in the choroid was found in both the membrane and cytosolic fractions and showed similar subcellular distribution as NOS activity, while endothelial (e) NOS protein in the choroid was present almost solely in the membrane fraction. Total NOS activity (nNOS + eNOS) and protein levels of nNOS and eNOS in the choroid were significantly reduced 6 weeks after the induction of diabetes. Conclusions. The high NOS activity in the choroid measured in vitro appears to come mostly from nNOS. Because choroidal nNOS exists in the parasympathetic perivascular nerve fibers, the decrease in choroidal nNOS in diabetic eyes suggests that the choroid undergoes a diabetes-induced neuronal disorder. Thus, diabetic choroidopathy encompasses diabetic neuropathy and microangiopathy.

Does precipitation reduce tissue staining by indocyanine green dye solutions

PURPOSE Indocyanine green (ICG) dye precipitates when mixed with certain ophthalmic irrigation solutions. The purpose of this study is to investigate whether this precipitation reduces ICG staining of the anterior lens capsule. METHODS ICG was diluted with each of the following solutions: BSS Plus, physiological saline, or Opeguard Neo. The products were then examined for green precipitate by light microscopy. The tissue staining capability of each ICG solution was tested at two different concentrations (0.5% and 0.0625%) in porcine lenses, regardless of whether the solution contained precipitate. RESULTS Green precipitate was observed in both concentrations of ICG solutions diluted with BSS Plus, but not in the solutions diluted with either physiological saline or Opeguard Neo. As assessed with light microscopy, staining of the anterior lens capsule appeared weaker for all 0.0625% ICG solutions compared to the corresponding 0.5% ICG solutions. The precipitate that formed in the 0.5% ICG solution diluted with BSS Plus had little effect on the staining quality of the anterior lens capsule. In contrast, the 0.0625% ICG solution diluted with BSS Plus (w/precipitate) showed weaker staining in the lens capsule compared to the other two 0.0625% ICG solutions (w/o precipitate). CONCLUSION These results suggest that precipitation of ICG may weaken its capability to stain the anterior lens capsule or other transparent ocular tissues. Therefore, ICG solutions that do not form a precipitate may be more capable of staining tissues at lower concentrations. As for other possibilities to explain the deterioration in staining, the effect of the composition of BSS Plus should also be considered.

Primary Conjunctival Follicular Lymphoma Treated with the Anti-CD20 Antibody Rituximab and Low-Dose Involved-Field Radiotherapy

reported. Although a PCR-determined monoclonal rearrangement of IgH in B cell lymphomas is strong evidence for the diagnosis of PIOL, there has been no other report of this fi nding. Only a few malignant cells were present in the vitreous cavity, and they were poorly preserved in the cases reported heretofore, so it was diffi cult to determine B cell monoclonality from both PIOL and PCNSL. It was diffi cult in our case, as well. In all these cases, including ours, the PCNSL was detected fi rst by brain biopsy and treated with radiation of the brain and systemic chemotherapy. In spite of the systemic chemotherapy, a PIOL developed later. We suggest that the tumor cells probably escaped the initial treatment because of their virulence, or the failure of systemic chemotherapy to contact the malignant cells due to the blood–ocular barrier. However, mild vitreous opacity existed prior to PCNSL in our case. Interestingly, bilateral identical monoclonality in PIOL has been detected without PCNSL involvement. This may indicate that tumor cells may have already have been present in the eye in this reported case. The fi nding of an identical IgH gene rearrangement supports the idea that the PCNSL and PIOL in our patient resulted from a dissemination of the same neoplastic clone rather than a simultaneous neoplastic formation of multiple B cell clones.