Structural and functional diversity of lysyl oxidase and the LOX-like proteins.

Lysyl oxidase (LOX) and four lysyl oxidase-like proteins, LOXL, LOXL2, LOXL3 and LOXL4, each contain a copper binding site, conserved lysyl and tyrosyl residues that may contribute to quinone co-factor formation, and a cytokine receptor-like domain. Each protein differs mainly in their N-terminal sequence, which may confer individual functions. Processing of the LOX proteins by BMP-1 and possibly other mechanisms may result in multiple functional forms. Splicing, reported for LOXL3, may also generate additional variants with unique functions. Each LOX, with its individual, developmentally regulated tissue and cell-specific expression and localization, results in a complex structural and functional variation for the LOX amine oxidases. The presence of only two LOX-like proteins in Drosophila, each with distinct spatial and temporal expression, allows for the assignment of individual function to one of these amine oxidases. Comparative expression analysis of each LOX protein is presented to help determine their functional significance.

Molecular and mutational analysis of a gelsolin-family member encoded by the flightless I gene of Drosophila melanogaster.

The flightless locus of Drosophila melanogaster has been analyzed at the genetic, molecular, ultrastructural and comparative crystallographic levels. The gene encodes a single transcript encoding a protein consisting of a leucine-rich amino terminal half and a carboxyterminal half with high sequence similarity to gelsolin. We determined the genomic sequence of the flightless landscape, the breakpoints of four chromosomal rearrangements, and the molecular lesions in two lethal and two viable alleles of the gene. The two alleles that lead to flight muscle abnormalities encode mutant proteins exhibiting amino acid replacements within the S1-like domain of their gelsolin-like region. Furthermore, the deduced intron-exon structure of the D. melanogaster gene has been compared with that of the Caenorhabditis elegans homologue. Furthermore, the sequence similarities of the flightless protein with gelsolin allow it to be evaluated in the context of the published crystallographic structure of the S1 domain of gelsolin. Amino acids considered essential for the structural integrity of the core are found to be highly conserved in the predicted flightless protein. Some of the residues considered essential for actin and calcium binding in gelsolin S1 and villin V1 are also well conserved. These data are discussed in light of the phenotypic characteristics of the mutants and the putative functions of the protein.

A novel human lysyl oxidase-like gene (LOXL4) on chromosome 10q24 has an altered scavenger receptor cysteine rich domain.

We have identified a novel 14-exon human lysyl oxidase-like gene, LOXL4, on chromosome 10q24. The cDNA and derived amino acid sequence of LOXL4 demonstrates a conserved C-terminal region including the characteristic copper-binding site, lysyl and tyrosyl residues and a cytokine receptor-like domain. One of the four N-terminal SRCR domains contains a 13 amino acid insertion encoded by a short exon not present within the closely homologous LOXL2 and LOXL3 genes. The 3.5-kb LOXL4 mRNA is present in pancreas and testis and at lower levels in several other tissues. Fibroblasts, smooth muscle and osteosarcoma (HOS) cells express LOXL4. No expression was detected in HCT-116 and DLD-1 colon, MCF-7 breast and DU-145 prostate cancer cell lines.

Intrathecal cyclosporin prolongs survival of late-stage ALS mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by upper and lower motor neuron death with ascending paralysis leading to death. In a transgenic mouse model of ALS (SOD1-G93A) weakness appears at 3 months of age, and because of progressive paralysis leads to death by 5 months. Cyclosporin A (CsA) is well known, for its extracerebral effect, as an immunosuppressant in organ transplantation. When able to access the brain, CsA is an effective neuroprotective agent mainly due to its protection of mitochondria through inhibition of the mitochondrial permeability transition. CsA does not cross the intact blood-brain barrier and was in the present study delivered to the brain through an infusion into the lateral cerebral ventricle. Injections started at the onset of late disease when weakness of the hindlimbs was apparent. CsA treatment prolonged the survival of ALS transgenic mice as compared to vehicle-treated controls. This finding implicates mitochondrial function in ALS and may have significance for human disease.

Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels.

The aims of this study were to (a) determine the prevalence of patients without elevated thyroid hormone levels in Graves' ophthalmopathy (GO) using current generation free thyroid hormone assays, (b) measure the prevalence of thyrotropin receptor antibodies (TRAb) in these cases, and (c) identify possible predictors of hyperthyroidism. Over a 30-month period, 1020 cases of thyroid eye disease were evaluated, of which only 19 (1.9%) met the diagnostic criteria. Ten (1%) had subclinical thyrotoxicosis, 7 (0.7%) were euthyroid, and 2 (0.2%) were hypothyroid as determined by a third-generation thyrotropin (TSH) assay. TRAb levels were measured in 16 of these 19 patients. The prevalence of TRAb varied according to the assay used. Polyethylene glycol-extracted thyroid-stimulating immunoglobulin (PEG-TSI), unfractionated thyroid-stimulating immunoglobulin (uTSI), first-generation porcine TSH-binding inhibitory immunoglobulin (pTBII), and second-generation human TSH-binding inhibitory immunoglobulin (hTBII) assays were positive in 93.8%, 50%, 18.8%, and 81.3% of patients, respectively. TRAb was detected by at least one method in all patients. Patients were followed up for 15 to 45 months. Hyperthyroidism developed in 4 patients (25%). Suppressed TSH levels and elevated TBII were predictors of hyperthyroidism. When sensitive assays are used, the prevalence of GO patients without elevated thyroid hormone levels is extremely low. The sensitivities of assays for TRAb detection differ substantially in these cases. PEG extraction improves the detection rate of TSI (p = 0.02), and hTBII assays improve the detection of TBII in these patients (p = 0.002). The high prevalence of TRAb in such cases supports a role for these antibodies in the pathogenesis of thyroid-associated eye disease.

The combination of absent thyroid peroxidase antibodies and high thyroid-stimulating immunoglobulin levels in Graves' disease identifies a group at markedly increased risk of ophthalmopathy.

Among Graves' Disease (GD) patients, we have observed an unexpectedly high prevalence of antithyroperoxidase antibody (TPOAb) and antithyroglobulin antibody (TgAb) negativity in those with severe ophthalmopathy. To study the possible role of thyroid autoantibodies in the pathogenesis of Graves' ophthalmopathy (GO), TPOAb, TgAb, thyroid-stimulating immunoglobulin (TSI), and thyrotropin-binding inhibitory immunoglobulin (TBII) levels were measured, and the presence or absence of GO was assessed by a single observer in 100 consecutive patients with newly diagnosed, untreated GD who were nonsmokers. Ophthalmopathy was present in 43 patients. TSI levels (p = 0.001), and the prevalence of TPOAb-negativity (p = 0.002) were significantly higher in patients with ophthalmopathy compared to those without. Logistic regression analysis showed that TSI levels (p = 0.005) and the absence of TPOAb (p = 0.0025) were independent predictors of GO. No correlation between TBII or TgAb and eye disease was found. The prevalence of GO increased with each quartile of TSI levels. The prevalence was 20%, 36%, 52%, and 64% in the first, second, third and fourth quartiles of TSI, respectively. The odds ratio of GO (with 95% confidence intervals) when TSI levels were above the median level (1640%) was 3.6 (1.5-8.0), when TPOAb was negative it was 5.0 (1.7-14.4), and with both risk factors it was 36.6 (4.3-313.5). The prevalence of ophthalmopathy in this last group was 92.9%. The combination of negative TPOAb and high TSI levels appears to be associated with a markedly increased risk of clinically evident ophthalmopathy.

Anti-Fas antibody differentially regulates apoptosis in Fas ligand resistant carcinoma lines via the caspase 3 family of cell death proteases but independently of bcl2 expression.

Deregulation of cell death pathways is an important feature of tumorigenesis. Fas, a member of the tumor necrosis factor receptor superfamily, is a transmembrane protein that can transduce cell death signals via a proteolytic cascade upon crosslinking or ligand binding. Fas has been implicated in the cell turnover of normal stratified squamous epithelia. To determine if altered Fas mediated cell death pathways participate in epithelial tumorigenesis, we examined squamous cell carcinoma (SCC) lines for sensitivity to Fas ligand (FasL) or an agonistic anti-Fas antibody. All cell lines examined were resistant to FasL mediated cell death. The carcinoma cell line SCC71 was also highly resistant to anti-Fas antibody. Another line, SCC9, underwent rapid cell death with characteristic features of apoptosis after exposure to anti-Fas antibody. However, binding of both FasL and anti-Fas antibody recruited downstream effector molecules to the Fas cytoplasmic domain in both SCC9 and SCC71 cells. Inhibition of the caspase 3- but not the ICE family of cell death proteases blocked apoptosis in SCC9 cells independently of expression of the anti-apoptotic protein bcl2. We concluded that Fas differentially mediates apoptosis in SCC lines by activation of caspase 3 family members but independent of bcl2 expression.

Infections of the eye.

Infections of the eye range from the mild to the severe. They may affect the lid, conjunctiva, cornea, lacrimal apparatus, vitreous, orbit or retina. Common presentations, diagnostic methods and treatment are briefly described. The doctor should not only manage outpatients appropriately, but know when to seek the advice of the ophthalmologist when indicated. Common conditions which can be managed by the general practitioner include blepharitis, styes, chalazia and conjunctivitis. With increased use of contact lenses, infection of the cornea has become more common. Sight-threatening conditions like endophthalmitis and orbital cellulitis should be recognised and treated early.

Hydroxyapatite orbital implants--our local experience.

Hydroxyapatite orbital implants were first introduced by Arthur Perry in 1985 and approved by the Food and Drug Administration for general use in 1989. Since its inception, it has become a popular orbital implant as it combines biocompatibility with improved motility. At the Singapore National Eye Centre, twenty consecutive patients underwent hydroxyapatite implantation from June 1993 to June 1996. Seventeen patients who had a follow-up time of more than 6 months were analysed retrospectively. Sixteen had enucleation while one had evisceration performed. The implant size used was either 18 or 20 mm. Seven primary and 10 secondary hydroxyapatite implants were performed in 17 patients ranging in age from 20 to 65 years. Six consisted of coralline hydroxyapatite while the remaining 11 were made of artificial hydroxyapatite. In the follow-up time of 6 to 16 months (mean 10.2 months), there have been no cases of infection, extrusion or migration. Two patients who had implant exposure were managed surgically with fascia lata grafts and have healed well. It appears that hydroxyapatite is the implant of choice for an anophthalmic socket. It becomes incorporated by the patients own tissues and once vascularised, it is less likely to migrate or extrude. The complication rate is low and implant exposure, if it occurs, can be easily managed.

Multiple evanescent white dot syndrome--an uncommon cause for an enlarged blind spot.

This report describes 3 cases of multiple evanescent white dot syndrome (MEWDS) which presented to the Singapore National Eye Centre over a 3-year period. MEWDS is an usual condition with distinct clinical features and typical findings on fundal fluorescein angiography. As the retinal lesions are transient, timing of examination is crucial for an accurate diagnosis. Usually only one eye is involved and recurrences are uncommon. Occasionally, signs of optic nerve disease may be the predominant feature. Although the aetiology remains unknown and no active treatment is required, the visual prognosis remains good.

Integrated hydroxyapatite implant and non-integrated implants in enucleated Asian patients.

INTRODUCTION: This study compares the outcome and complications of integrated hydroxyapatite implant and non-integrated orbital implants following enucleation in Asian patients. MATERIALS AND METHODS: This is a retrospective study of enucleated patients with coralline hydroxyapatite implants versus non-integrated implants (acrylic, glass and silicone) at the Singapore National Eye Centre from January 1991 to December 2000. The outcomes measured were implant migration, extrusion, socket infection, conjunctival dehiscence and implant exposure. Statistical analysis was done using the 2-sample t-test. RESULTS: Twenty-one patients had the hydroxyapatite implant and 38 non-integrated implants (27 acrylic, 9 glass and 2 silicone). The mean duration of follow-up was 2.7 years and 4 years for the hydroxyapatite implant and non-integrated implants respectively. Three patients with pre-existing severe socket contracture before enucleation surgeries were excluded from the study. Four cases of implant migration, 4 cases of implant extrusion and 3 cases of socket infection were encountered; all were sockets fitted with non-integrated implants. There was a higher rate of conjunctival dehiscence for sockets with hydroxyapatite implants (6 out of 21) compared to sockets with non-integrated implants (3 out of 35). This was statistically significant (P = 0.048). CONCLUSIONS: Implant complications of migration, extrusion and socket infection were found in non-integrated implants and none in coralline hydroxyapatite implants, which had a significantly higher rate of conjunctival dehiscence. Most of these were easily managed with only a small number progressing to implant exposure.

Using the phacoemulsification crescent knife in dacryocystorhinostomy.

Dacryocystorhinostomy is an effective treatment for nasolacrimal duct obstruction. Most techniques employ a conventional blade or knife in making the incision of the nasal mucosa and lacrimal sac. The authors describe the use of a phacoemulsification crescent knife for this purpose. This technique can be effective and at the same time safer and easier to perform.

Active lysyl oxidase (LOX) correlates with focal adhesion kinase (FAK)/paxillin activation and migration in invasive astrocytes.

The extracellular matrix (ECM) plays a critical role during the development and invasion of primary brain tumours. However, the function of ECM components and signalling between a permissive ECM and invasive astrocytes is not fully understood. We have recently reported the ECM enzyme, lysyl oxidase (LOX), in the central nervous system and observed up-regulation of LOX in anaplastic astrocytoma cells. While the catalytic function of LOX is essential for cross-linking of ECM proteins, we also reported that LOX induced invasive and metastatic properties in breast tumour epithelial cells through hydrogen peroxide-mediated FAK/Src activation. In this study, we tested the hypothesis that active LOX is expressed in anaplastic astrocytes and promotes FAK activation and invasive/migratory behaviour. Results demonstrate that increased expression and activity of LOX positively correlated with invasive phenotype of malignant astrocytoma cell lines. Immunohistochemistry detected increased LOX within tumour cells and ECM in grade I-IV astrocytic neoplasm compared with normal brain and coincidence of increased LOX with the loss of glial fibrillary acidic protein in higher-grade tumours. Increased active LOX in invasive astrocytes was accompanied by phosphorylation of FAK[Tyr576] and paxillin[Tyr118]; furthermore, both FAK and paxillin tyrosine phosphorylation were diminished by beta-aminopropionitrile inhibition of LOX activity and depletion of H(2)O(2) via catalase treatment. Additionally, we provide evidence that in astrocytes, LOX is likely processed by bone morphogenic protein-1 and LOX activity might be further stimulated by the expression of fibronectin in these cells. These results demonstrate an important LOX-mediated mechanism that promotes migratory/invasive behaviour of malignant astrocytes.

An unusual clinical presentation of angular vein varix.

Varices of the angular vein may arise anywhere along its length that may give rise to different clinical presentations. We report a case of a 56 year old male who presented with a medical canthal mass of 6 months duration. Surgical exploration and excison biopsy showed varix of the angular vein.

Congenital upper lid colobomas: management and visual outcome.

PURPOSE: Congenital upper lid colobomas may be associated with ocular and systemic anomalies. The ophthalmologist's role involves both the diagnosis and management of their various ocular structural defects and management of the visual development. METHODS: All cases of congenital eyelid colobomas referred to the Singapore National Eye Centre between July 1992 and July 1995 were assessed for the extent of the eyelid defect, associated ocular anomalies, status of the cornea, and any systemic anomalies. RESULTS: Four infants were tested during this period. The mean follow-up was 25 months (range, 16 to 30 months). All required surgical correction of their eyelid defects before 2 years of age. The patients also had refractive errors requiring amblyopia management. Three of the babies also needed other surgical procedures. CONCLUSIONS: Congenital upper eyelid colobomas are a potential threat to vision at an early age and a significant cosmetic blemish later in life. Early surgical intervention is usually required when the defect is larger than one third of the eyelid margin. Close monitoring of the visual development of patients with congenital upper eyelid colobomas is also essential since the risk of amblyopia in these patients is high.

Clinical and microbial spectrum of fungal keratitis in Singapore: a 5-year retrospective study.

BACKGROUND: The epidemiology of fungal keratitis varies geographically, but commonly occurs in warm, tropical climates. To determine the microbial and clinical characteristics of this disease in Singapore, we conducted a 5-year hospital-based retrospective study. METHODS: A retrospective review of culture-positive fungal keratitis at the Singapore National Eye Center and Singapore General Hospital, from January 1991 to December 1995. RESULTS: Twenty-nine consecutive cases of culture-positive fungal keratitis were seen over the study period. The mean age of the cases was 41 years and 23 were males. Amongst the varied occupations, 9 were construction workers. The most common cultured organisms were Fusarium sp. (52%) and Aspergillus flavus (17%). More than half had a history of ocular trauma prior to the development of keratitis, while a quarter had antecedent topical corticosteroid therapy. In contrast, only 2 patients were contact-lens wearers. Despite medical therapy, 10 patients eventually required therapeutic penetrating keratoplasties; of these, 6 were caused by Fusarium species. CONCLUSION: Fusarium is the commonest cultured organism in fungal keratitis in Singapore and is associated with significant ocular morbidity.

Surgical management of upper lid epiblepharon.

BACKGROUND: Upper lid epiblepharon refers to a developmental anomaly characterised by the presence of an exaggerated skin fold running horizontally across the upper lid below the lid margin, which in severe cases is associated with lash-cornea touch or even corneal punctate epithelial erosions. METHOD: This retrospective study looked at 14 patients who underwent bilateral upper lid pretarsal orbicularis debulking and skin excision to correct lash-globe contact from the upper lid. RESULTS: Surgery was successful in 25 of 28 lids, with one recurrence being successfully re-operated. The remaining 2 recurrences did not undergo further surgery as the patients were asymptomatic. CONCLUSION: This surgical procedure appears to be an effective treatment of lash inversion in upper lid epiblepharon with good aesthetic results.

Novel proteins interacting with the leucine-rich repeat domain of human flightless-I identified by the yeast two-hybrid system.

The flightless-I gene encodes a member of the gelsolin-like family of actin-binding proteins linked to a leucine-rich repeat (LRR) domain. It is required for cellularization during early embryogenesis and normal development of the indirect flight muscles in Drosophila melanogaster. Although the association between actin and the gelsolin-like domain of the human Flightless-I homologue (FLI) has been established, its biological role is unknown. The human FLI gene is mapped within the Smith-Magenis microdeletion region of chromosome 17. We report the identification of two related genes, LRRFIP1 and LRRFIP2, encoding proteins that interact with the LRR domain of human FLI using the yeast two-hybrid system. LRRFIP1 exhibits sequence identity with the TRIP RNA-binding protein and GCF-2 transcriptional repressor, which are also related to the murine FLAP-1 gene. LRRFIP2 is a novel gene that shares sequence homology with LRRFIP1 and FLAP-1. LRRFIP1 and LRRFIP2 both express alternative splice variants in heart and skeletal muscle tissue. A coiled-coil domain, conserved within each encoded protein, serves as a potential interaction motif for FLI LRR. The occurrence of multiple proteins able to interact with FLI within the same tissue suggests that they may compete for the same binding site. Sequencing and PCR-directed genomic analysis indicate that LRRFIP1 and LRRFIP2 are related genes that arose from gene duplication.

Risk factors and clinical outcomes between fungal and bacterial keratitis: a comparative study.

PURPOSE: Previous studies on fungal and bacterial keratitis were descriptive single case series analysis. We conducted a hospital-based retrospective study to evaluate fungal and bacterial keratitis using a case-control design to compare risk factors and clinical outcomes. METHODS: Twenty-nine cases of culture-positive fungal keratitis seen over a 5-year period were compared to 51 cases of culture-positive bacterial keratitis seen over a 21 months period. Using bacterial keratitis as the reference group, case-control odds ratios (OR) for predisposing factors and cohort relative risks (RR) for clinical outcomes associated with fungal keratitis were derived. Mantel-Haenszel adjustment procedures were used to examine the respective roles of confounding and intermediate variables. RESULTS: Compared to bacterial keratitis, fungal keratitis was significantly more likely to be associated with ocular trauma (OR = 2.69, 95% confidence interval [CI], 1.06-6.86) but significantly less likely to be associated with contact lens wear (OR = 0.16, 95% CI, 0.04-0.67) and preexisting ocular diseases (OR = 0.23, 95% CI, 0.07-0.72). Fungal keratitis was more likely to perforate than bacterial keratitis (RR = 5.28, 95% CI, 1.35-20.66) and to require penetrating keratoplasty (OR = 5.86, 95% CI, 2.06-16.69). CONCLUSIONS: Fungal keratitis appears more likely to result from ocular trauma, whereas bacterial keratitis is more likely to result from contact lens wear and pre-existing ocular diseases. Fungal keratitis is more likely than bacterial keratitis to result in perforation and require penetrating keratoplasty.

Refractive change following pterygium surgery.

PURPOSE: We conducted a prospective study of patients with primary pterygia to analyze surgically-induced astigmatic changes following pterygium surgery and their relation to pterygium morphology and size. METHODS: One hundred twenty-three eyes of 123 adult patients with primary pterygia were evaluated. Pterygium was graded according to morphology and the extent of corneal encroachment. Manifest refraction was performed preoperatively and at 1, 3, and 6 months postoperatively. Patients underwent either the bare sclera technique of pterygium excision or conjunctival grafting. Surgery was performed by one surgeon. Olsen's method of vector decomposition was used to analyze surgically-induced astigmatism. RESULTS: The mean magnitude of preoperative astigmatism was 0.99 D, with vector decomposition revealing a relative flattening of the cornea in the horizontal meridian. Astigmatism of 1.00 D or more was seen when the pterygium exceeded 3.5 mm beyond the limbus. Postoperatively, a steepening of the cornea in the horizontal meridian was demonstrated, the magnitude of which was related to pterygium size (P = 0.0001). ANOVA testing showed no significant difference (P > 0.05) when pterygia were divided according to morphology. CONCLUSIONS: This study confirms that pterygium excision induces a reversal of pterygium-related corneal flattening. A strong correlation was also found between the horizontal extent of pterygium encroachment and astigmatic change following surgery.