Formation of an intermediate compound with a B12H12 cluster: experimental and theoretical studies on magnesium borohydride Mg(BH4)2.

Experimental and theoretical studies on Mg(BH4)2 were carried out from the viewpoint of the formation of the intermediate compound MgB12H12 with B12H12 cluster. The full dehydriding and partial rehydriding reactions of Mg(BH4)2 occurred according to the following multistep reaction: Mg(BH4)2 -->1/6MgB12H12 + 5/6MgH2 + 13/6H2 <--> MgH2 + 2B + 3H2 <--> Mg + 2B + 4H2. The dehydriding reaction of Mg(BH4)2 starts at approximately 520 K, and 14.4 mass% of hydrogen is released upon heating to 800 K. Furthermore, 6.1 mass% of hydrogen can be rehydrided through the formation of MgB12H12. The mechanism for the formation of MgB12H12 under the present rehydriding condition is also discussed.

A fixative suitable for in situ hybridization histochemistry.

We compared the morphology and stability of hybridization signals between paraffin sections of rat retina fixed with commonly used 4% paraformaldehyde/PBS and those fixed with a fixative containing glutaraldehyde in in situ hybridization histochemistry, using a digoxigenin-labeled RNA probe complementary for beta-galactoside alpha 2,6-sialyltransferase mRNA. Retinal detachment was frequently observed in the sections fixed with 4% paraformaldehyde-PBS, whereas the morphology was satisfactorily preserved in those fixed with either 0.5% glutaraldehyde, 4% paraformaldehyde-PBS, or 2.5% glutaraldehyde-PBS. Without glutaraldehyde, it was difficult to determine the most appropriate length of proteinase K digestion of tissue sections for facilitating probe penetration, since the optimal time for definite hybridization was variable among the retinal cells in heterogeneous layers. By addition of glutaraldehyde to paraformaldehyde or with glutaraldehyde alone, it was easy to establish the appropriate time for the unmasking procedure, since intense mRNA signals were constant throughout the retina by proteinase K digestion for more than 30-40 min. Using a fixative that causes stronger cross-linking (e.g., glutaraldehyde) is recommended to improve not only the morphology but also the stability of hybridization signals in in situ hybridization histochemistry with paraffin embedding and digoxigenin-labeled RNA probes.

Isolation and characterization of mucinlike glycoprotein associated with photoreceptor cells.

PURPOSE: Although previous lectin-histochemical studies have shown that O-linked glycoproteins are distributed in cone pedicles and rod spherules, as well as in photoreceptors, including associated interphotoreceptor matrices (IPM), attention has been directed only to those in the IPM. In this study, cloning of the O-linked glycoproteins not only in the IPM but also in the region including the cone pedicles and rod spherules was attempted. METHODS: The cDNA for the core protein of the O-linked glycoprotein in the bovine retina was isolated by screening a bovine retinal cDNA library using a polyclonal antibody against the jacalin (a lectin specific for O-linked sugar residues)-binding glycoproteins (JBGPs) in the whole bovine retina. The expression of the JPGP core protein in the retina was examined by means of in situ hybridization histochemistry and immunohistochemistry. RESULTS: The cDNA was isolated and found to encode an entire core protein [predicted molecular mass (Mr): 101 kDa; rich in Ser and Thr; mucin-like] for the JBGPs with Mr of 120 and 135 kDa. The mRNA was expressed in both cone and rod photoreceptor cells. This protein was distributed in the cone pedicles and rod spherules as well as the photoreceptor layer. CONCLUSIONS: Mucinlike glycoproteins with Mr of 120 and 135 kDa may be synthesized in the cone and rod photoreceptor cells, respectively, and distributed not only in the photoreceptor layer (probably including the IPM) but also in the cone pedicles and rod spherules.

Isolation and characterization of galectins in the mammalian retina.

PURPOSE: Previous studies have suggested that galectins may be involved in retinal adhesion and photoreceptor cell survival. To elucidate the underlying mechanisms, the authors isolated retinal galectins, determined their types and distributions, and investigated the validity of the hypothesis, using rat models. METHODS: An antibody was prepared against a bovine retinal lectin that was isolated by use of a lactose-agarose column. cDNA of the lectin was isolated by screening of a bovine retinal cDNA library, using the antibody, and then was sequenced. The cDNAs of rat retinal galectins were also isolated by means of polymerase chain reaction and used to produce an antibody against recombinant galectin-3. Using the described antibodies, the authors examined the distributions of galectins in bovine and rat retinas, morphologic changes of rat retinas induced by the antibodies, and distributional changes of galectins in constant-light-exposed rat retinas. RESULTS: The cDNAs of bovine galectin-1, rat galectin-1, and rat galectin-3 were isolated. Galectin-1 was found in various regions, including the retinal pigment epithelium, outer limiting membrane, and outer plexiform layer in bovine and rat retinas. Galectin-3 was increasingly detected in the cytoplasm of Müller cells after constant light exposure after an increase in its transcript. Retinal detachment and vacuolation of the outer plexiform layer were induced in rat eyes by intravitreous injection of the anti-galectin-1 antibody. CONCLUSIONS: Galectin-1 may be involved in adhesion of the photoreceptor and outer plexiform layers by interacting with glycoconjugates with beta-galactoside residues in the interphotoreceptor matrix and synaptic cleft matrix. Galectin-3 may increase in Müller cells of a degenerative rat retina, probably through endogenous anti-apoptosis.

Functional Rescue of photoreceptors from the damaging effects of constant light by survival-promoting factors in the rat.

PURPOSE: To investigate whether and how survival-promoting agents rescue photoreceptor cell function and morphology from constant light damage, the authors recorded electroretinographic (ERG) responses and examined light micrographs of retinas in those rats given intravitreal injection of midkine (MK) and basic fibroblast growth factor (bFGF) before constant exposure. METHODS: Albino Sprague-Dawley rats were injected with MK, bFGF, or phosphate-buffered saline (PBS) 2 days before the onset of 1 week of constant light. ERG responses were recorded using white flash stimuli with the intensity range of 4 log units, followed by histologic examinations of retinas, including quantitative assessment of the outer nuclear layer thickness as an index of photoreceptor cell loss. RESULTS: ERG responses were barely detectable in uninjected eyes after 1 week of constant light. On the other hand, distinct responses were recordable in eyes injected intravitreally with MK and bFGF, and the degree of ERG rescue in terms of the amplitude of b-wave was approximately 40% to 60% compared with normal eyes. Intravitreally injected PBS showed slight, but noticeable, preservation of ERG responses as well. Histologic examination revealed that MK and bFGF protected photoreceptors from light damage. A good correlation was found between anatomic rescue of photoreceptors as assessed by outer nuclear layer thickness and the functional rescue as defined by the magnitude of ERG responses. CONCLUSIONS: Functional and anatomic rescue of photoreceptors in albino rats from constant light damage is achieved by prior intravitreal injection of MK and bFGF.

Rescue of photoreceptors from the damaging effects of constant light by midkine, a retinoic acid-responsive gene product.

PURPOSE: To evaluate the protective effects of midkine (MK), the product of a retinoic acid-responsive gene, on constant light-induced retinal degeneration in albino Sprague-Dawley rats. METHODS: Midkine, basic fibroblast growth factor (bFGF), MK plus heparin, or buffer controls were injected intravitreally 2 days before constant light exposure. After 7 days of continuous light exposure, the eyes were perfused with fixative, bisected along the vertical meridian, embedded in paraffin, and sectioned. The degree of retinal light damage was assessed for paraffin-embedded sections by cytologic analysis, by measuring the thickness of the outer nuclear layer (ONL), and by counting the number of macrophages. RESULTS: After 1 week of constant light exposure, uninjected controls and those injected with phosphate-buffered saline (PBS) lost most of the photoreceptor inner and outer segments, and the thickness of the ONL was decreased. Eyes that were injected with MK or bFGF demonstrated a significant rescue in the photoreceptor layer with a two- to threefold increase in the ONL thickness. The number of macrophages in eyes injected with MK was significantly suppressed compared with controls. Those injected with bFGF had a 1.5-fold increase in number compared with controls. CONCLUSIONS: Midkine has shown strong survival-promoting activity in constant light-induced retinal degeneration, and thus has a high degree of neurotrophic activity in vivo.

Initial stages of posterior vitreous detachment in healthy eyes of older persons evaluated by optical coherence tomography.

OBJECTIVE: To promote understanding of the development of posterior vitreous detachment (PVD) in healthy eyes using optical coherence tomography (OCT). METHODS: We studied 209 eyes of 209 healthy volunteers (165 men and 44 women; mean age, 52.3 years [range, 31-74 years]). In addition to biomicroscopy and ophthalmoscopy, OCT was performed to obtain high-resolution cross-sectional images of the vitreoretinal interface in the posterior fundus. RESULTS: The condition of the posterior vitreoretinal interface was classified as 1 of 5 stages, according to biomicroscopic findings and OCT images relative to discrete linear signals indicating a detached posterior vitreous face: stage 0, no PVD (61 eyes [29.2%]); stage 1, incomplete perifoveal PVD in up to 3 quadrants (100 eyes [47.8%]); stage 2, incomplete perifoveal PVD in all quadrants, with residual attachment to the fovea and optic disc (26 eyes [12.4%]); stage 3, incomplete PVD over the posterior pole, with residual attachment to the optic disc (4 eyes [1.9%]); or stage 4, complete PVD identified with biomicroscopy, but not with OCT because of instrument limitations (18 eyes [8.6%]). Stage 1, 2, and 3 incomplete PVD without subjective symptoms was not recognizable on contact lens biomicroscopy. There was a significant age-related progression in the condition of the vitreoretinal interface from stage 0 to stage 4. The superior quadrant was usually the initial site of incomplete PVD. CONCLUSIONS: Optical coherence tomography demonstrates that healthy human eyes have incomplete or partial PVD beginning as early as the fourth decade of life. Age-related PVD occurs initially as a focal detachment in the perifovea of 1 quadrant, with persistent attachment to the fovea and optic nerve head, with a predilection for the superior quadrant. It extends its range slowly for years and eventually results in complete PVD, associated with release of vitreopapillary adhesion.

Postsurgical evaluation of idiopathic vitreomacular traction syndrome by optical coherence tomography.

PURPOSE: To report a case of idiopathic vitreomacular traction syndrome with preoperative and postoperative evaluation by optical coherence tomography. DESIGN: Interventional case report. METHODS: A 62-year-old woman presented with blurred vision in the left eye because of idiopathic vitreomacular traction syndrome, and she underwent a pars plana vitrectomy. Optical coherence tomography was performed before and after surgery. RESULTS: Preoperative optical coherence tomography, right eye, revealed residual adhesion of incomplete posterior vitreous detachment and edematous, thickened outer retina in the macula. A successful vitrectomy relieved vitreoretinal traction with nearly complete resolution of cystoid macular edema within 1 month after surgery, followed in subsequent months by gradual foveal depression resembling a lamellar macular hole. Resolution of subretinal serous fluid was delayed with complete disappearance, some 12 months after surgery, which correlated with a gradual improvement in visual acuity. CONCLUSION: Optical coherence tomography provides a sensitive anatomical evaluation of vitreomacular traction syndrome. Reorganization of retinal tissue after surgical intervention for vitreoretinal traction may be slower than is apparent from conventional examinations.

Retinopathy associated with Machado--Joseph disease (spinocerebellar ataxia 3) with CAG trinucleotide repeat expansion.

PURPOSE: To report characteristic atrophic maculopathy in a patient with Machado--Joseph disease (spinocerebellar ataxia 3) caused by trinucleotide repeat expansion of the relevant gene. METHODS: Case report. RESULTS: A 64-year-old Japanese man had suffered from slurred speech and gait disturbance since 57 years of age. Cerebellar ataxia, extensor plantar response, and other neurological signs were compatible with features of Machado--Joseph disease. Magnetic resonance imaging showed atrophies of cerebellum and cerebral cortex. Family history suggested an autosomal dominant inheritance of the disease. The patient presented with gaze-evoked nystagmus and limitations of eye movement in all directions. Ophthalmoscopy and fluorescein angiogram revealed symmetric changes in the posterior fundi, which consisted of patchy atrophies at the level of the retinal pigment epithelium. Scotopic electroretinogram showed no abnormalities with normal oscillatory potentials. Polymerase chain reaction analysis of the Machado--Joseph disease gene identified a heterozygous trinucleotide (CAG) repeat expansion. CONCLUSION: This case illustrates a rare association of atrophic maculopathy and external ophthalmoplegia in Machado--Joseph disease, contrasted with the common occurrence of retinal degeneration in spinocerebellar ataxia 7. Dystrophic changes in the retinal pigment epithelium have rarely been described but may be one of the characteristic complications of Machado--Joseph disease.

Genetic association of manganese superoxide dismutase with exudative age-related macular degeneration.

PURPOSE: To elucidate whether any polymorphic genes for xenobiotic-metabolizing and antioxidant enzymes are associated with the development of exudative age-related macular degeneration. METHODS: A hospital-based case-control study was performed on a consecutive series of 102 Japanese patients with the exudative form of age-related macular degeneration who were recruited between 1993 and 1998 in the Kagoshima University Hospital. Controls were 200 systemically healthy individuals who had no senescent ocular disorders and were over 50 years of age. There was no evidence of age-related macular degeneration in the 200 controls. Genomic DNA from peripheral bloods was examined using polymerase chain reaction and defined for the genetic polymorphisms of cytochrome P-450 1A1, glutathione S-transferases, microsomal epoxide hydrolase, and manganese superoxide dismutase. RESULTS: We found a significant association of manganese superoxide dismutase gene polymorphism, valine/alanine polymorphism at the targeting sequence of the enzyme, with age-related macular degeneration. The patients had an increased frequency of alanine allele and alanine/alanine genotype (odds ratio = 10.14, 95% confidence interval = 4.84 to 2.13; P =.0005 after Bonferroni correction). We also observed a weak association of microsomal epoxide hydrolase exon-3 polymorphism with age-related macular degeneration (odds ratio = 2.20, 95% confidence interval = 4. 02 to 1.20; P =.020 after Bonferroni correction). Cytochrome P-450 1A1, glutathione S-transferases, and microsomal epoxide hydrolase exon-4 were polymorphic, but their genotype frequency distributions did not show a statistically significant difference between the patients and controls. CONCLUSIONS: The results suggest that manganese superoxide dismutase gene polymorphism is associated with exudative age-related macular degeneration. Microsomal epoxide hydrolase is another enzyme that may be associated with the disease. The exudative form of age-related macular degeneration may have genetic risk factors against oxidative stress and/or effects of xenobiotics. Further association studies in other polymorphic genes for xenobiotic-metabolizing enzymes are needed to elucidate the environmental-genetic interaction in the underlying cause of age-related macular degeneration.

A novel truncating mutation of cytochrome P4501B1 (CYP1B1) gene in primary infantile glaucoma.

PURPOSE: To report a novel mutation of the CYP1B1 gene in a Japanese patient with primary infantile glaucoma. METHODS: DNA was extracted from leukocytes of six unrelated patients with primary infantile glaucoma. The coding regions of the CYP1B1 gene were amplified by polymerase chain reaction, examined by agarose gel separation and heteroduplex methods, and directly sequenced. RESULTS: One of the patients with primary infantile glaucoma had a mutation of the CYP1B1 gene, with an abnormal shift in agarose gel separation and heteroduplex analysis. Direct sequencing disclosed a homozygous insertion of guanine at nucleotide 1620 of exon 3, which produced a frameshift leading to premature termination of amino acid translation. The patient was male and had sporadic, classic primary infantile glaucoma. None of the other five patients with infantile glaucoma, the 30 patients with primary adult-onset glaucoma, or the 70 healthy control subjects showed any abnormalities in the CYP1B1 gene. CONCLUSIONS: This is the first report of CYP1B1 gene mutation in primary infantile glaucoma from the Eastern world. CYP1B1 gene mutation for primary infantile glaucoma spreads worldwide, but its prevalence may have ethnic or geographic differences.

Cytochrome P450 1B1 gene mutations in Japanese patients with primary congenital glaucoma(1).

PURPOSE: To report a novel missense mutation and DNA polymorphism of the CYP1B1gene in Japanese patients with primary congenital glaucoma. METHODS: A series of 11 unrelated patients with primary congenital glaucoma was examined. Patients were followed in the Kagoshima University Hospital between 1979 and 1998. DNA was extracted from leukocytes of the patients, their families, and unrelated healthy individuals. Amplicons spanning the coding regions of the CYP1B1 gene were examined by direct sequencing and enzyme-restriction detection. RESULTS: In the 11 unrelated patients, besides the previously reported insertional mutation (1620 ins G), a novel missense mutation was identified at codons 444 to replace arginine with glutamine (R444Q) in one patient. The novel missense mutation cosegregated in the relevant family as an autosomal recessive pattern and was not found in other patients or control individuals. In addition, five polymorphic sites were found at codons 48, 119, 330, 432, and 449. These polymorphic alleles did not cosegregate with the disease, and they were found in healthy individuals as well. CONCLUSIONS: Approximately 20% of Japanese patients with primary congenital glaucoma may be affected by mutations in the CYP1B1 gene. Further studies are justified to explore whether a relationship exists between the phenotypic expressivity of the disease and the type of mutation.

Hepatocyte growth factor stimulates cell growth and enhances the expression of transforming growth factor alpha mRNA in AsPC-1 human pancreatic cancer cells.

We investigated the effects of hepatocyte growth factor (HGF) and transforming growth factor alpha (TGF alpha) on cell growth in four human pancreatic cancer cell lines. Changes in the expression of mRNAs of HGF, c-met, TGF alpha, and epidermal growth factor receptor (EGFR) by treatment with HGF and TGF alpha were observed. Cell growth with growth factors was assessed with the MTT assay and compared with basal growth without growth factors. Although HGF stimulated cell growth in AsPC-1, COLO-357, and T3M4 cells, Panc-1 cells showed no response to HGF. TGF alpha stimulated the growth of all the above cells. The expression of c-met mRNA under nonstimulated conditions was detected with Northern blotting in all cells. Treatment with HGF slightly enhanced the expression of c-met mRNA only in COLO-357 cells. The intensity of EGFR expression was consistent, and HGF mRNA was not detected during induction experiments in any cell type. Concomitant treatment with HGF and TGF alpha exerted an effect that was additive or less on the growth of all cells. Expression of TGF alpha was enhanced by HGF treatment only in AsPC-1 cells. These results suggested that HGF and TGF alpha stimulated cell growth through a final common pathway of signal transduction.

Corneal lesions induced by the systemic administration of capsaicin in neonatal mice and rats.

Following a single subcutaneous injection of capsaicin to neonatal mice, a high incidence of corneal lesions with opacity developed after a long latency. The intensity of the lesions progressed for about 1 month in animals which had received a high dose (50 or 100 mg/kg) of capsaicin. Although the intensity gradually decreased thereafter, 50% of animals still exhibited a visible opacity 6 months after treatment. Similar corneal lesions were also produced in neonatal rats which had been injected with capsaicin. It is suggested that the corneal lesions induced by capsaicin may be due to destruction of the trigeminal nerve.

Cloning, expression and sequence analysis of cDNA for the luciferases from the Japanese fireflies, Pyrocoelia miyako and Hotaria parvula.

Cloning and sequence analysis of cDNA for the luciferases of Pyrocoelia miyako and Hotaria parvula were carried out (GenBank accession numbers L39928 and L39929, respectively). The amino acid sequence, deduced from the nucleotide sequence, showed P. miyako luciferase to consist of 548 amino acid residues with a molecular weight of 60,955, while the luciferase of H. parvula consisted of 548 amino acid residues with a molecular weight of 60,364. Pyrocoelia miyako luciferase showed 82.1% homology with the luciferase of Photinus pyralis and less than 70% homology with other firefly luciferases, whereas H. parvula luciferase showed 98%, 82.5% and 81.2% homology with the luciferases of Luciola mingrelica, Luciola lateralis and Luciola, cruciata respectively. Two regions in the enzymes were found to be highly conserved. The amino acid sequences were used to construct a phylogenetic tree, which showed that the fireflies could be divided into two groups.

Primary vitreoretinal dysplasia resembling Norrie's disease in a female: association with X autosome chromosomal translocation.

A female infant with the typical clinical and histopathological features of vitreoretinal dysplasia is described. She had an apparently balanced reciprocal chromosomal translocation 46XX,t(X;10) with the X chromosome breakpoint being on the short arm. Since the parents' karyotypes were normal, it is most plausible that a de novo chromosomal translocation disrupted the vitreoretinal dysplasia gene itself. The severe clinical symptoms of this heterozygous female patient were explained by non-random X inactivation. She may have had Norrie's disease, an X linked recessive disorder due to an X autosome translocation.

Clinical features of HTLV-I associated uveitis.

The prevalence of human T cell lymphoma virus type 1 (HTLV-I) was studied among patients with endogenous uveitis. Twelve (15.8%) of 76 uveitis patients with known aetiology or clinical entity were seropositive, the prevalence being comparable with that in the general population of the southwestern area of Japan where HTLV-I is highly endemic. In the comparison, 32 (41.0%) of 78 patients with aetiology or entity undefined uveitis were seropositive for HTLV-I, which indicated a significantly higher seroprevalence than controls matched for sex and age. The 32 cases of clinical entity undefined, HTLV-I positive uveitis were characterised by acute granulomatous or non-granulomatous uveal reactions which were accompanied by vitreous opacities and retinal vasculitis. The uveal inflammatory and retinal vascular changes responded well to topical and/or systemic corticosteroids and resolved in a few weeks in the majority of cases with favourable visual outcome. The disease affected one or both eyes, and eight cases (25%) showed recurrence within a year. The general condition of the patients remained well otherwise during a follow up study (mean follow up time 15.4 months), except for three cases with a possible association of hyperthyroidism. These findings provide additional information favouring an association between HTLV-I and isolated uveitis, a new disease entity which should be termed HTLV-I-associated uveitis.

HTLV-I associated uveitis revisited: characteristic grey-white, granular deposits on retinal vessels.

AIMS: To elucidate whether there exists any clinical sign characteristic of HTLV-I associated uveitis. METHODS: Fifty five patients with HTLV-I associated uveitis were reviewed. These cases had serum antibodies to HTLV-I, and any other uveitis entities were carefully excluded by means of clinical and laboratory studies. RESULTS: Eight cases (14.5%) developed vascular lesions in the retina, characterised by grey-white, granular deposits scattered on the retinal veins and/or arteries in the posterior pole. The vascular changes did not accompany any haemorrhage, sheathing, or leakage of fluorescent dye on angiograms, and the retina was otherwise unremarkable. A single or clustered form of similar materials was also found to deposit on the vitreo-retinal interface of the foveolar area. These deposits resolved in a few weeks spontaneously or in response to corticosteroids together with anterior uveal inflammation. CONCLUSION: The vascular lesions described here suggest a characteristic sign for HTLV-I associated uveitis, and it may provide, if recognised, an additional clinical marker to establish diagnosis.

Fundus flavimaculatus: polymorphic retinal change in siblings.

A 12-year-old boy and an 11-year-old girl, siblings of healthy, consanguineous parents, had a bilateral retinal dystrophy with a gradual loss of vision. The brother showed a bull's eye macular change with sparse fundus flavimaculatus type flecks. The sister had numerous fleck lesions of fundus flavimaculatus throughout the posterior fundus, but there was virtually no macular change. Thus the siblings presented instances of polymorphic expressivity of fundus flavimaculatus.

Seroprevalence of antibodies to HTLV-I in patients with ocular disorders.

Human T-lymphotropic virus type 1 (HTLV-I) has been shown to spread worldwide and to be responsible for distinct systemic diseases, namely adult T-cell leukaemia and HTLV-I-associated myelopathy. Immune-mediated, inflammatory lesions in the lungs, joints, and lacrimal glands (Sjögren's syndrome) are also suggested to be associated with the retrovirus. We studied seroprevalence of antibodies to HTLV-I in patients with various ocular disorders who are residents of south-west Japan, one of the endemic areas of HTLV-I. Of 310 patients with ocular disease 72 (23.2%) were seropositive. This seroprevalence did not differ significantly from that of the general population of the area. As regards individual ocular diseases, aetiologically undefined nonspecific uveitis showed a significantly high seropositivity for HTLV-I. Of 44 patients 18 (40.9%) were seropositive. Their clinical features were acute or subacute, transient and sometimes recurrent, and granulomatous changes in the anterior uvea. Patients with isolated cotton-wool spot of the retina, non-familial retinitis pigmentosa, or keratoconjunctivitis sicca did not show any significantly high prevalence of HTLV-I infection.