Identification of the gene responsible for gelatinous drop-like corneal dystrophy.

Gelatinous drop-like corneal dystrophy (GDLD; OMIM 204870) is an autosomal recessive disorder characterized by severe corneal amyloidosis leading to blindness, with an incidence of 1 in 300,000 in Japan. Our previous genetic linkage study localized the gene responsible to a 2.6-cM interval on chromosome 1p. Clinical manifestations, which appear in the first decade of life, include blurred vision, photophobia and foreign-body sensation. By the third decade, raised, yellowish-grey, gelatinous masses severely impair visual acuity, and lamellar keratoplasty is required for most patients. Here we report DNA sequencing, cDNA cloning and mutational analyses of four deleterious mutations (Q118X, 632delA, Q207X and S170X) in M1S1 (formerly TROP2 and GA733-1), encoding a gastrointestinal tumour-associated antigen. The Q118X mutation was the most common alteration in the GDLD patients examined, accounting for 33 of 40 (82.5%) disease alleles in our panel of families. Protein expression analysis revealed aggregation of the mutated, truncated protein in the perinuclear region, whereas the normal protein was distributed diffusely in the cytoplasm with a homogenous or fine granular pattern. Our successful identification of the gene that is defective in GDLD should facilitate genetic diagnosis and potentially treatment of the disease, and enhance general understanding of the mechanisms of amyloidosis.

Variability of parvovirus B19 genotype 2 in plasma products with different compositions in the inactivation sensitivity by liquid-heating.

BACKGROUND AND OBJECTIVES: Our previous report showed that parvovirus B19 genotype 1 in different solutions derived from plasma preparations showed different heat-sensitivity patterns during liquid-heating. In this study, we similarly examined B19 genotype 2. MATERIALS AND METHODS: Two plasma samples one containing B19 genotype 1 and the other genotype 2 DNA were used. Four process samples collected immediately before the heat treatment step in the manufacture of albumin, immunoglobulin, haptoglobin and antithrombin preparations were spiked with B19 and subsequently treated at 60°C for 10 h. A low pH immunoglobulin solution was also spiked with B19 and treated at room temperature for 14 days. Infectivity was then measured. RESULTS: B19 genotype 2, similar to genotype 1, showed three patterns of inactivation: (i) a rapid inactivation in the albumin and immunoglobulin preparations, (ii) a slow inactivation in the haptoglobin preparation and (iii) only limited inactivation in the antithrombin preparation. Its sensitivity in the low pH immunoglobulin solutions also resembled that of genotype 1. CONCLUSION: Both genotypes 1 and 2 of B19 varied in sensitivity to liquid-heating and low pH among different plasma preparations.

Reliability and validity of Japanese version of the McGill Quality of Life Questionnaire assessed by application in palliative care wards.

The McGill Quality of Life Questionnaire (MQOL), which consists of 16 items constructing physical, psychological, existential and support subscales and one item of overall quality of life (QOL), has been developed to assess QOL of terminal cancer patients. To examine if MQOL Japanese version (MQOL-J) is applicable, it was administered to 83 terminal cancer patients in palliative care wards several days after admission and then 7 to 10 days after the first interview. Cronbach's alpha coefficient for four subscales was 0.584-0.860. Sixteen items were classified into four factors by factor analysis, similar to the original English version. The results indicated that psychological and existential domains of the MQOL-J significantly related to overall QOL. Existential and support domains as well as overall QOL were significantly improved between the first and second interviews, although performance status assessed by Eastern Cooperative Oncology Group worsened. It is suggested that MQOL-J can reflect perceived health status of terminal cancer patients.

Electronic structure and magnetic anisotropy of a constrained Fe chain in an electric field.

The electronic structure and magnetic anisotropy were studied for a constrained iron chain in an electric field by means of the first-principles approach. The electron induced by the electric field was found to occupy mainly the extended s-like orbitals which is well hybridized with the d(3z(2)-r(2)) localized orbital. The magnetic anisotropy was observed to decrease with the number of induced electrons and to depend on the magnetization direction. The magnetization perpendicular both to the chain and the electric field modifies the electron dispersion relation to be asymmetrical, which implies an expectation of variable transport property with both the external electric and magnetic fields.

Extent of hepatitis E virus elimination is affected by stabilizers present in plasma products and pore size of nanofilters.

BACKGROUND AND OBJECTIVE: To investigate the physico-chemical properties of hepatitis E virus (HEV) with regard to inactivation/removal, we have studied four isolates with respect to sensitivity to heat during liquid/dry-heating as well as removal by nanofiltration. MATERIALS AND METHODS: Hepatitis E virus in an albumin solution or phosphate-buffered saline (PBS) was liquid-heated at 60 degrees C for a preset time. HEV in a freeze-dried fibrinogen containing stabilizers was also dry-heated at 60 or 80 degrees C for a preset time. In addition, to clarify the removal of HEV, the purified virus in PBS was filtered using several types of virus-removal filter (nanofilters) that have different pore sizes. HEV infectivity or genome equivalents before and after the treatments were assayed by a semiquantitative cell-based infectivity assay or quantitative polymerase chain reaction assay, respectively. RESULTS: Hepatitis E virus isolates in albumin solutions were inactivated slowly at 60 degrees C for 5 h and the resultant log reduction factor (LRF) was from 1.0 to > or = 2.2, whereas the virus in PBS was inactivated quickly to below the detection limit and the LRF was > or = 2.4 to > or = 3.7. The virus in a freeze dried fibrinogen containing trisodium citrate dihydrate and l-arginine hydrochloride as stabilizers was inactivated slowly and the LRF was 2.0 and 3.0, respectively, of the 72 h at 60 degrees C, but inactivated to below the detection limit within 24 h at 80 degrees C with an LRF of > or = 4.0. The virus in PBS was also confirmed as to be approximately 35 nm in diameter by nanofiltration. These results are useful for evaluating viral safety against HEV contamination in blood products. CONCLUSION: The sensitivity of HEV to heat was shown to vary greatly depending on the heating conditions. On the other hand, the HEV particles were completely removed using 20-nm nanofilters. However, each inactivation/removal step should be carefully evaluated with respect to the HEV inactivation/removal capacity, which may be influenced by processing conditions such as the stabilizers used for blood products.

Intravitreal injection of bevacizumab for choroidal neovascularisation associated with pathological myopia.

AIM: To assess the efficacy and safety of an intravitreal injection of bevacizumab (Avastin(R)) for myopic choroidal neovascularisation (mCNV). METHODS: Intravitreal bevacizumab (1 mg) was injected into eight eyes of eight patients with mCNV in this non-randomised, interventional case series. The best-corrected visual acuity (BCVA) was measured and the optical coherence tomography (OCT) and fluorescein angiography findings were examined before and after treatment. The minimum follow-up time was 3 months. RESULTS: The mean BCVA was 0.26 before treatment and 0.51 at the last visit (p = 0.009). The BCVA improved to two or more lines in six eyes (75%) and remained the same in two eyes (25%). Leakage from the mCNV on fluorescein angiography decreased in seven eyes (87.5%). The choroidal neovascularisation area on fluorescein angiography (p = 0.049) and the foveal thickness on OCT images decreased significantly (p = 0.027) after the treatment. No major complications developed. CONCLUSION: Intravitreal injection of bevacizumab seems to be an effective and safe treatment for mCNV.

Magnetic anisotropies of iron on the Pt(111) surface.

We have studied magnetic anisotropies of Fe atoms on the platinum (111) surface, employing a fully relativistic pseudopotential and plane wave method with the local spin density approximation. We investigated three surface structures with different Fe monolayer coverages: full coverage, half-coverage and quarter-coverage. The effect of surface relaxation has been included. It was found that the magnetic easy axis of the system is within the surface plane for all systems investigated. In the system of an Fe chain on Pt(111), having an anisotropic local structure, the magnetic anisotropy energy is much enhanced after surface relaxation. This absolute value is larger compared with the value for bulk alloy and the magnetic easy axis is directed parallel to the alignment of Fe atoms.

Secretory production of recombinant urokinase-type plasminogen activator-annexin V chimeras in Pichia pastoris.

To produce a thrombi-targeting plasminogen activator, we expressed a fused gene that contains a modified pre-sequence of Mucor pussilus rennin (MPR) followed by a chimeric gene of single-chain urokinase-type plasminogen activator (scu-PA)::annexin V (AV). The fused gene was ligated into an integrative vector, under the control of the alcohol oxidase 1 (AOX1) promoter (p), and transformed into Pichia pastoris. Transformants were monitored for the secretion of fibrinolytic activity. The highest expressing clone, HB225, secreted as much as 600 international units (IU) of fibrinolytic activity per ml of culture medium under optimal conditions. It contained three tandem copies of the full-size vector disruptively integrated into the AOX1 sequence. Western blot analysis revealed that the secreted chimera was highly susceptible to proteolysis. Addition of excess amino acids (aa) to the culture medium minimized the degree of proteolysis. Two major species of chimera, 85 and 65 kDa, were then isolated from the culture medium. The former was the intact form consisting of a single-chain and showing full enzyme activity after activation by plasmin. The latter was an enzymatically processed form consisting of two chains held by a disulfide bond, having full enzyme activity without activation. Both chimeras exhibited calcium-dependent phospholipid (PL)-binding affinities similar to the parent AV.

Rapid detection of M1S1 mutations by the protein truncation test.

PURPOSE: To determine a method of rapid detection of M1S1 gene mutations in patients with gelatinous droplike corneal dystrophy. METHODS: Forty-one patients from 35 families with gelatinous drop-like corneal dystrophy were studied. The entire coding region of the M1S1 gene was screened using the protein truncation test (PYT), with a polymerase chain reaction fragment amplified from genomic DNA serving as a template of in vitro translation. RESULTS: Homozygous or compound heterozygous mutations were detected in all patients by a single reaction of the PTT. This result matched those obtained using the polymerase chain reaction-restriction fragment length polymorphism and direct sequence analyses. The Q118X mutation was present in 63 of the 70 alleles, accounting for 90% of the disease-associated chromosomes in Japanese patients. CONCLUSIONS: The PTT is useful for detecting mutations in the M1S1 gene. This technique showed that the Q118X mutation is a founder mutation in Japanese patients with gelatinous droplike corneal dystrophy, and it reflects the linkage disequilibrium reported previously.

The effect of TGF-beta1 on differential gene expression profiles in human corneal epithelium studied by cDNA expression array.

PURPOSE: TGF-betas regulate cell proliferation and differentiation, and they play important roles in maintenance of corneal epithelium. However, the precise function of TGF-betas in the corneal epithelium remains unclear. In this study, cDNA expression array technology was used to demonstrate the effect of TGF-beta1 on the simultaneous expression of a large number of genes in cultured human corneal epithelial cells (HCECs). The change in protein level expression of the specific genes influenced by TGF-beta1 was also investigated. METHODS: Human cDNA expression array technology was used to study the simultaneous expression of 1176 specific cellular genes in HCECs incubated with TGF-beta1 (10 ng/ml). Moreover, gene-specific semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to confirm the gene expression pattern measured by the cDNA expression array. Western blot analysis was used to examine protein expression of the specific genes in the presence or absence of TGF-beta1. RESULTS: TGF-beta1 significantly upregulated the expression of 19 genes and significantly downregulated ras-related protein, caspase10, and beta4-integrin in the treated HCECs. The expression of 277 genes including alpha3-integrin, PAI-2, transferrin receptor, and cyclin-D1 was studied. Semiquantitative RT-PCR analysis confirmed the TGF-beta1-mediated changes in expression patterns of these genes. Furthermore, Western blot analysis revealed that TGF-beta1 remarkably decreased PAI-2, transferrin receptor, and integrin alpha3, and increased caspase10 on the protein level. CONCLUSIONS: TGF-beta1 regulates the expression of specific types of genes in HCECs. These results strongly suggest that TGF-beta1 is critically involved in the maintenance of the corneal epithelium through the control of a network of various signal-transduction pathways.

X-linked retinoschisis with point mutations in the XLRS1 gene.

BACKGROUND: X-linked retinoschisis (XLRS) is a relatively rare vitreoretinal dystrophy that causes visual loss in young men. Recently, a gene responsible for this disease, designated XLRS1, was identified, and several deleterious gene mutations were reported. OBJECTIVE: To analyze Japanese patients clinically diagnosed as having XLRS formutational changes in the XLRS1 gene. METHODS: Ten patients with XLRS underwent full ophthalmologic examination, including slitlamp biomicroscopy and dilated funduscopy. Genomic DNA was isolated from leukocytes, and all exons of the XLRS1 gene were amplified by polymerase chain reaction and analyzed using a direct sequencing method. RESULTS: Point mutations in the XLRS1 gene were identified in all 10 patients. The mutations were identical in each of 2 pairs of brothers. Six of the point mutations represented missense mutations, 1 was a nonsense mutation, and 1 was a frameshift mutation. Five of the mutations are newly reported herein. CONCLUSIONS: The discovery of new point mutations in this study increases the available information regarding the spectrum of genetic abnormalities and clinical manifestations of XLRS. However, the limited data failed to reveal a correlation between mutation and disease phenotype. CLINICAL RELEVANCE: Identification of mutations in the XLRS1 gene and expanded information on clinical manifestations will facilitate early diagnosis, appropriate early therapy, and genetic counseling regarding the prognosis of XLRS.

Two patterns of opacity in corneal dystrophy caused by the homozygous BIG-H3 R124H mutation.

PURPOSE: To investigate the opacity pattern in corneas with an Arg124His (R124H) homozygous mutation of the BIG-H3 gene. METHODS: Slit-lamp examination was performed on eight patients with corneal dystrophy resulting from a genetically confirmed BIG-H3 R124H homozygous mutation. The birthplace of each patient also was determined. RESULTS: Slit-lamp examination disclosed two types of opacity patterns in corneas with the BIG-H3 R124H homozygous mutation. Type I (n = 4) is a spot-like opacity present in the anterior stroma in which the lesions are confluent. Type I is the same pattern that previous reports have shown to be caused by the BIG-H3 R124H homozygous mutation. The type II corneal opacity pattern (n = 4) is a reticular opacity in the anterior stroma with round translucent spaces. Type II opacity has not been reported previously in association with any corneal dystrophy. The patients with the type I opacity do not share a common birthplace; however, interestingly, the patients with the type II opacity traced their origin to Tottori prefecture in western Japan. CONCLUSION: The BIG-H3 homozygous R124H mutation induces the development of two distinct patterns of corneal opacity, the recognition of which can establish an accurate diagnosis of corneal dystrophy caused by the homozygous BIG-H3 R124H mutation independent of genetic analysis. In addition, genetic factors or circumstantial influences other than the gene responsible for the corneal dystrophy may influence the pattern of corneal opacity.

Two distinct kerato-epithelin mutations in Reis-Bücklers corneal dystrophy.

PURPOSE: Two patients were diagnosed with Reis-Bücklers corneal dystrophy (RBCD), although the pattern and severity of corneal opacification differed. To see whether there was a genetic basis for these phenotypic variations, we analyzed beta ig-h3, the gene that codes for kerato-epithelin and that contains a mutation (Arg555Gln) that causes RBCD. METHODS: A 30-year-old man with honeycomb-shaped subepithelial opacities in his central cornea and a 25-year-old man with progressive subepithelial geographic opacities were both considered to have RBCD. We isolated genomic DNA from leukocytes of the two patients and their family members and screened for an Arg555Gln kerato-epithelin mutation. Then we analyzed all exons of the gene using the single-strand conformation polymorphism (SSCP) technique to search for any other kerato-epithelin mutations. RESULTS: The patient with honeycomb-shaped opacities had an Arg555Gln kerato-epithelin mutation that caused his RBCD, whereas the patient with geographic opacities did not; instead, he had a new kerato-epithelin mutation (Arg124Leu), which cosegregated with his family members. CONCLUSIONS: The variant of RBCD characterized by honeycomb-shaped opacities is caused by an Arg555Gln kerato-epithelin mutation. On the other hand, a new kerato-epithelin mutation, Arg124Leu, was found to cause the RBCD variant characterized by recurrent epithelial erosions and progressive geographic subepithelial opacification. Codon 124 is a hot spot for kerato-epithelin mutations, where the mutations responsible for three autosomal dominant corneal dystrophies--lattice type I (Arg124Cys), Avellino (Arg124His), and the variant of RBCD with geographic rather than honeycomb opacities (Arg124Leu)--are located.

Granular corneal dystrophy with homozygous mutations in the kerato-epithelin gene.

PURPOSE: To report a family with several members affected with granular corneal dystrophy Groenouw type 1. Three members of the family were affected with a severe placoid type of corneal dystrophy. To determine the relationship between gene mutations and phenotypic variations of the disease, we analyzed the kerato-epithelin gene. METHODS: The pedigree included a consanguineous marriage of two affected individuals. The three family members affected with a severe form of corneal dystrophy were offspring of these parents. However, the phenotype of other affected family members was typical granular corneal dystrophy. We isolated genomic DNA from leukocytes of the family members. Exons of the keratoepithelin gene were amplified by the polymerase chain reaction and were analyzed using the single-strand conformation polymorphism technique. Mutations were identified by direct sequencing method and restriction digestion analysis. RESULTS: The three severely affected family members exhibited homozygous mutations at codon 555 (arginine to tryptophan) in the keratoepithelin gene, whereas those with typical granular corneal dystrophy had the heterozygous mutation at the same codon. Unaffected family members did not have the mutation. CONCLUSIONS: We determined that the severe phenotype of granular corneal dystrophy is caused by homozygous mutations in the kerato-epithelin gene. Clinical manifestation of the severe phenotype is a placoid type of corneal dystrophy and early recurrence after surgery. Granular corneal dystrophy appears to be the first ophthalmic disease in which homozygosity for a dominant allele has been genetically identified.

Chorioretinal damage caused by the excision of choroidal neovascularization.

PURPOSE: To determine whether choroidal neovascularization excision causes mechanical damage to the neurosensory retina, retinal pigment epithelium, or choriocapillaris. METHODS: Prospectively, 18 eyes of 18 consecutive patients who underwent choroidal neovascularization excision were observed. Preoperatively and postoperatively, the integrity of the choriocapillaris circulation in the pathway of choroidal neovascularization extraction was studied by fluorescein and indocyanine green angiography. Using static scanning laser ophthalmoscope microperimetry, the presence of iatrogenic scotomas that developed postoperatively in the pathway of choroidal neovascularization extraction was also investigated. RESULTS: Postoperatively, a choriocapillaris defect was detected in 17 (94.4%) of 18 cases. In 15 cases (83.3%), the choriocapillaris defect had a clear relationship to the pathway of choroidal neovascularization extraction. Postoperatively, a scotoma was present in 16 (88.9%) of 18 cases. In 14 cases (77.8%), the location of the scotoma had a clear relationship to the pathway of choroidal neovascularization extraction. CONCLUSION: Surgical excision of choroidal neovascularization leads to severe damage of the choroid and retina in the pathway of the extracted choroidal neovascularization. The injury involves the neurosensory retina, retinal pigment epithelium, and choriocapillaris.

Differentiating full thickness macular holes from impending macular holes and macular pseudoholes.

AIMS: The reliability of scanning laser ophthalmoscope (SLO) microperimetry in differentiating full thickness macular holes from macular pseudoholes and impending macular holes was evaluated. METHODS: 106 eyes with the clinical diagnosis of full thickness macular holes, macular pseudoholes, and impending (stage 1) macular holes were examined for the presence of deep or relative scotoma using SLO microperimetry. The relation between these scotomas and the clinical diagnosis was studied. RESULTS: Deep and relative scotomas were detected in all 57 eyes with clinically defined full thickness macular holes. In contrast, among 49 eyes diagnosed with macular pseudoholes or impending macular holes, no deep and only one relative scotoma was observed. The sensitivity of the presence of a deep scotoma as an indicator of the clinical diagnosis of a full thickness macular hole was 100% (57 of 57), and the specificity was 100% (49 of 49). The sensitivity of the presence of a relative scotoma was 100% (57 of 57) and the specificity was 98.0% (48 of 49). CONCLUSION: With SLO microperimetry, full thickness macular holes can be precisely and objectively distinguished from other conditions that mimic macular holes.

The spectrum of beta ig-h3 gene mutations in Japanese patients with corneal dystrophy.

PURPOSE: This study was undertaken to identify beta ig-h3 gene mutations in Japanese patients with granular corneal dystrophy (GCD), Avellino corneal dystrophy (ACD), lattice corneal dystrophy (LCD), and Reis-Bücklers' corneal dystrophy (RBCD). R124H, R124C, R555W, and R555Q mutations have been reported in Europe to cause ACD, LCD type I, GCD, and RBCD, respectively. METHODS: In total, 91 Japanese patients who had been clinically diagnosed with GCD, LCD, or RBCD were investigated to determine whether they had mutations in the beta ig-h3 gene. Genomic DNA was amplified using the polymerase chain reaction and analyzed using single-strand conformation polymorphism techniques. Mutations were identified using the direct sequencing method. RESULTS: In 68 unrelated patients who had been diagnosed with GCD, 62 patients (91%) were found to have the R124H mutation, which has been reported to cause ACD, whereas only six patients (9%) had the R555W mutation. In LCD patients, 10 patients with type I disease had the R124C mutation, and 10 patients with type IIIA disease had a P501T mutation. One patient with atypical LCD had an L527R mutation. In two patients with RBCD, one had an R555Q mutation and the other patient with geographic opacities was found to have an R124L mutation. CONCLUSIONS: Depending on the specific mutation in the beta ig-h3 gene, the phenotypes of corneal dystrophy may differ. Our results indicate that assay of mutations in the beta ig-h3 gene is required to establish a correct diagnosis.

Effect of early feeding on cellularity of rat adipose tissue.

Effect of food intake at various ages (fetal, suckling and weaning periods) on adipose tissue cellularity was studied. 1) Rats were given a restricted diet on weaning and later allowed free access to diet. Fat pads of these animals recovered normal weight on refeeding and no differences in cell number or cell size were observed. 2) Food intake was varied by changing the litter size during the suckling period and, after weaning, animals were given free access to diet. Fat pads of rats raised in small litters were heavier than those of rats raised in large litters. The differences in adipose tissue weight were accounted for by differences in cell size and cell number 3) Maternal rats were given a restricted diet during pregnancy. The pups were irregular in their weight. Pups of the restricted group which were smaller than pups of the control group at birth were chosen and raised normally. Fat pads of these animals were lighter than those of control animals and could be explained in terms of differences in cell size. From these findings, it was suggested that cellular effects of early feeding depended on the phase of growth in the rat.

Changes in lipid synthesis in rat adipose tissue during development.

Lipogenesis in rat adipose tissue during postnatal development has been studied by measuring the incorporation of labeled glucose into lipid in fat cells and the activities of certain enzymes which participate in lipogenesis. It was found that: (1) Fatty acid synthesis from glucose is negligible in the suckling period. After weaning, it increases rapidly and reaches a plateau at the age of 10 weeks. (2) Insulin sensitivity is negligible in the suckling period. After weaning it rises and then declines. (3) Activities of ATP citrate lyase and acetyl CoA carboxylase are maintained at a low level in the suckling period. After weaning, these activities rise rapidly and then decline. (4) Weaning rats onto a high-fat diet showed decreases in glucose incorporation into fatty acids, insulin sensitivity, ATP citrate lyase activity and acetyl CoA carboxylase activity.

Effect of litter size on glucose metabolism in rat pup adipose tissue during the suckling period.

The effect of milk intake on the glucose tolerance capacity, lipogenesis, ATP citrate lyase and acetyl CoA carboxylase activities in rat adipose tissues during the suckling period was studied. The glucose tolerance capacity in the suckling rats was far lower than that in rats at the age of 5 weeks. However, glucose tolerance capacity in the suckling animals of 4-membered litters at the age of 2 weeks was higher than that in control animals of 8-12 membered litters at the same age. Lipid synthesis, fatty acid synthesis and ATP citrate lyase activity in subcutaneous adipose tissues of animals of 4-membered litters in the suckling period were higher than those of control animals. It was observed that ATP citrate lyase activity in subcutaneous adipose tissues of animals of 4-membered litters in the suckling period were higher than those of control animals. It was observed that ATP citrate lyase activity in adipose tissues during the suckling period was increased by glucose injection alone. These results suggest that the development of the glucose tolerance capacity and the lipogenesis in adipose tissues during the suckling period were influenced by nutritional status as well as chronological age.